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WO1998008860A1 - Nouvelles sequences polynucleotidiques codant des proteines impliquees dans la myogenese - Google Patents

Nouvelles sequences polynucleotidiques codant des proteines impliquees dans la myogenese Download PDF

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Publication number
WO1998008860A1
WO1998008860A1 PCT/US1997/014780 US9714780W WO9808860A1 WO 1998008860 A1 WO1998008860 A1 WO 1998008860A1 US 9714780 W US9714780 W US 9714780W WO 9808860 A1 WO9808860 A1 WO 9808860A1
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Prior art keywords
polynucleotide
mfa
seq
protein
expression
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PCT/US1997/014780
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English (en)
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C. M. Amy Chen
Norbert Kraut
Mark Groudine
Harold WEINTRAUB
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Fred Hutchinson Cancer Research Center
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Priority to AU40826/97A priority Critical patent/AU4082697A/en
Publication of WO1998008860A1 publication Critical patent/WO1998008860A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • NOVEL POLYNUCLEOTIDE SEQUENCES ENCODING PROTEINS INVOLVED IN MYOGENESIS
  • presomitic mesoderm primordial cells flanking the neural tube and notochord, called the presomitic mesoderm, mature into early segmental structures called the somites.
  • the presomitic mesoderm buds into segments to form the epithelial somites, this process proceeds in a cranio-caudal direction according to an intrinsic developmental timetable (reviewed by Keynes and Stern, Development 103: 413-429, 1989; Tarn and Traynor, Anat. Embrvol. 189: 275-305, 1994).
  • the ventral-medial parts of the somites delaminate to form the sclerotome, while the dorsal-lateral component of the somite forms the dermomyotome.
  • the fate of cells in the epithelial somites is plastic, or 'Undetermined," whereas after this stage the sclerotome cells are 'tommitted” to differentiate into the vertebral column and ribs, and dermomyotome cells are committed to form der is and axial skeletal muscle.
  • a key inductive signal in this regard may be provided by the gene Sonic hedgehog, which has been shown to enhance sclerotomal marker expression and repress dermomyotomal marker expression when the gene is expressed ectopically or in heterologous cells (Johnson et al., CeH 79: 1165-1173, 1994; Fan et al., £eM 79: 1175-1186, 1994).
  • MyoD family of myogenic factors which appear to act 'downstream" (i.e., subordinately in a developmental regulatory hierarchy) of Sonic hedgehog in determining somite cell fate.
  • the MyoD family of genes includes myoD, my ⁇ , myogenin and MRF4, which each encode muscle specific transcriptional regulatory factors belonging to the basic-helix-loop-helix (bHLH) class of DNA binding proteins (see reviews by Emerson, Curr. Biol. 2: 1065-1075, 1990; Weintraub, et al., Science 251: 761-766, 1991).
  • AU of the MyoD family of myogenic factors share the remarkable property of being able to convert cells into a myogenic differentiation pathway when the cells are transfected with a MyoD family member gene.
  • primary fibroblasts of different species transfected with the myoD gene are induced to express muscle specific genes, and in many cases form muscle fibers and differentiate into myoblasts or yotubes (see for example, Weintraub et al., Proc. Natl. Acad. Sci. USA 86: 5434-5438, 1989).
  • the myogenic activity of the MyoD family genes is explained in part by their conservative, bHLH domains, which includes a basic region required for DNA binding, and an HLH region required for dimerization (see for example, Davis et al., £___ 60: 733-746, 1990). Further explanation for the myogenic activity of MyoD family genes includes their ability to heter odimerize with 'E proteins" and the ability of these heterodimeric complexes to bind to the ⁇ box" sequence motif, CANNTG, of many muscle specific genes and transactivate their expression (reviewed by Kadesh, Imm. Today H: 31-36, 1992).
  • the invention achieves these objects and other objects and advantages which will become apparent from the description which follow.s by providing novel polynucleotide sequences, including a novel mammalian gene, Inhibitor of MyoD family (I-mf), and a number of corresponding cDNAs encoding myogenic regulatory proteins, I-mfa, I-mfb and I- mfc.
  • I-mf-encoding polynucleotides and regulatory proteins provided herein possess novel structural, biochemical and cell biological properties rendering these polynucleotides and proteins useful in the study and/or control of a variety of myogenic regulatory processes.
  • the I-mf polynucleotides and proteins disclosed herein are useful for generating, isolating, and characterizing additional endogenous regulatory factors, as well as I-mf analogs, drugs and other agents useful for evaluating and/or controlling myogenic processes in normal and abnormal vertebrate development and myogenesis.
  • the invention provides several methods and tools specifically directed to the diagnosis and treatment of disease conditions involving defective myogenic processes in mammals. Ithin one aspect of the invention, purified and isolated polynucleotides are provided that encode a native I-mf protein. Examples of these polynucleotides include three, full- length I-mf cDNA clones isolated from murine embryonic cDNA libraries.
  • polynucleotides encoding native I-mf proteins are provided that have variations in amino acid sequence compared to the exemplary murine cDNA clones described herein, such as variations due to allelic variations and genetic differences between species.
  • native I-mf-encoding polynucleotides hybridize under moderate to high stringency conditions to one or more of the aforementioned cDNAs, and encode biologically active products having at least 80% amino acid identity with one or more of the native murine I-mf proteins, I-mfa, I-mfb and I-mfc, or with a corresponding functional domain or peptide fragment of I- mfa, I-mfb and I-mfc.
  • I-mf analog-encoding polynucleotides that encode I-mf analogs.
  • I-mf analogs include genetically engineered, synthetic or otherwise artificially derived polynucleotides that hybridize under moderate to high stringency conditions to one or more of the aforementioned native cDNAs and encode functional I-mf analogs having at least 80% amino acid identity with one or more of the native murine I-mf proteins, I-mfa, I-mfb and I-mfc, or with a corresponding functional domain or peptide fragment of I-mfa, I-mfb and I-mfc.
  • I-mf analogs include mutant I-mf proteins generated by recombinant or synthetic methods, peptide fragments of I-mf, fusion proteins and the like incorporating only a portion of a native I-mf protein, and up to a full length, native I-mf protein.
  • Preferred I-mf analog-encoding polynucleotides in this context encode I-mf analogs possessing normally functional or hyperfunctional biological activity compared to a corresponding native I-mf protein, functional domain or peptide fragment.
  • I-mfa analog-encoding polynucleotides are provided that encode I-mf analogs having functional or hyperfunctional anti-myogenic activity compared to the anti-myogenic activity of corresponding native I-mfa in an in-vitro or in-vivo assay.
  • Particularly preferred I-mf analog-encoding polynucleotides encode I-mf analogs comprising discrete functional domains of a native I-mf protein, for example an I-mfa carboxyl terminal interactional domain ('I-mfa interactional domain'), which is sufficient to mediate interactions with the MyoD family of myogenic factors.
  • Alternative preferred embodiments within this aspect of the invention include mutant polynucleotides that encode hypofunctional analogs of I-mf proteins, or of selected functional domains of I-mf such as the I-mfa interactional domain, which exhibit impaired, null or hypomorphic biological activity compared to native I-mf proteins or I-mf functional domains.
  • polynucleotide expression constructs are provided for transforming or transfecting a suitable host cell to express an I-mf protein or I- mf analog.
  • These expression constructs include a polynucleotide expression vector operably coupled with a polynucleotide encoding an I-mf protein or I-mf analog to form an expression construct capable of directing the expression of the I-mf protein or I-mf functional analog by the host cell following introduction of the expression construct into the cell.
  • Exemplary I-mf based polynucleotide expression constructs of the invention incorporate a cDNA encoding I-mfa, I-mfb or I-mfc.
  • I-mf analog-encoding polynucleotides which hybridize under moderate to high stringency conditions to one or more of the aforementioned cDNAs encoding native I-mfa, I-mfb or I- mfc.
  • Suitable vectors for use within the polynucleotide expression constructs of the invention include DNA plasmid, DNA viral expression vectors, RNA viral expression vectors and the like.
  • the expression vector includes one or more elements that facilitate or control expression of I-mf protein or I-mf functional analog from the construct, such as an inducible promoter or enhancer sequence, a selectable marker sequence, a heterologous reporter sequence, a sequence encoding a retroviral transacting factor or the like.
  • methods are provided for expressing an I-mf protein or I-mf analog in a host cell.
  • a suitable host cell is selected, and a polynucleotide expression construct incorporating an I-mf based polynucleotide encoding an I-mf protein or I-mf analog is introduced into the host cell.
  • the expression construct is operable to transform the host cell by directing expression of the I-mf protein or I-mf functional analog in the cell.
  • the cell thus transformed is incubated in a suitable incubation medium under physiological conditions to promote expression of the I-mf protein or I-mf analog by the cell.
  • the I-mf protein or I-mf analog is isolated from the host cell or conditioned medium after its expression by the cell.
  • I- mf based expression constructs are employed in methods for modulating (i.e. enhancing or inhibiting) expression or activity of endogenous MyoD family proteins or I-mf proteins in host cells transformed or transfected with the construct.
  • I-mf based expression constructs are used in methods to induce ectopic expression of I-mfa or an I- mfa analog thereby inhibiting a myogenic activity of a MyoD family gene or its expression product.
  • the I-mf based expression construct is introduced into a selected host cell by microinjection, transformation, transfection, electroporation or other suitable method to achieve introduction of the construct into the host cell.
  • the transformed or transfected host cell is cultured, transplanted or left in situ under suitable conditions to permit the host cell to express the I-mf protein or I-mf analog.
  • Suitable host cells for use within these methods include a variety of known and readily available fungal, bacterial and mammalian cells routinely selectable to express the I-mf proteins and I-mf analogs of the invention.
  • transformed host cells transformed according the above methods are provided as novel compositions of matter, and as useful tools within yet additional methods of the invention, described herein.
  • substantially pure, native I-mf proteins including full length I-mfa, I-mfb and I-mfc, and products of interspecific homologs and intraspecific allelic variants of the I- f gene, are provided.
  • I-mf analogs including mutant I-mf proteins generated by recombinant or synthetic methods, peptide fragments of I-mf, fusion proteins and the like incorporating only a portion of a native I-mf protein and up to a full length, native I-mf protein.
  • Preferred I-mf analogs include functional or hyperfunctional I-mf analogs possessing detectable biological activity of a native I-mf protein.
  • I-mfa analog-encoding polynucleotides are provided that encode I-mf analogs having detectable anti-myogenic activity in an in-vitro or in-vivo assay.
  • These anti- myogenically active I-mf analogs alone or in the form of a fusion protein, exhibit one or more detectable anti-myogenic activities, which include: binding to a myogenic factor, inhibiting nuclear localization of a myogenic factor, inhibiting DNA binding of a myogenic factor, inhibiting transcriptional activation of muscle specific genes by a myogenic factor, and inhibiting myogenic differentiation of muscle phenotypes in mammalian cells.
  • Particularly preferred anti-myogenic I-mf analogs comprise discrete functional domains of a native I-mf protein, for example the I-mfa interactional domain.
  • Alternative preferred embodiments within this aspect of the invention include hypofunctional analogs of I-mf proteins, or of selected functional domains of I-mf such as the I-mfa interactional domain, which exhibit impaired, null, or hypomorphic anti-myogenic activity compared to native I-mf proteins or I- mf functional domains.
  • mutant analogs of I-mf proteins, or of selected functional domains of an I-mf protein, such as the I-mfa interactional domain, which exhibit modified biological activity compared to native I-mf proteins or I-mf functional domains are also provided within this aspect of the invention.
  • immunoreagents such as antibodies, antibody derivatives, chimeric antibodies and antibody conjugates are provided that bind specifically to one or more of the native I-mf proteins, peptides, and I-mf analogs disclosed herein.
  • exemplary immunoreagents include labeled antibodies, antibody derivatives and chimeric antibodies that bind specifically to one or more of the native I-mf proteins and I-mf analogs disclosed herein, thereby providing labeled probes to facilitate detection of antibody-I-mf complexes in a sample suspected of containing I-mf.
  • the immunoreagents of the invention can be used within the methods of the invention as effective tools for detecting and/or quantifying the expression, localization and/or activity of I-mf proteins, peptides or I-mf analogs, and can be incorporated in a wide variety of assays and screening methods disclosed herein.
  • anti-I-mf immunoreagents can themselves be used as agonists or antagonists of I-mf activity, for example by interfering with binding between I-mf and a myogenic factor such as MyoD or myf5 and thereby preventing or reducing anti-myogenic activity of I-mf in the presence of the antibody.
  • anti-I-mf antibody conjugates can be used as targeting agents for delivery of compounds of therapeutic interest.
  • immunoreagents provided and used within the invention include monoclonal antibodies, which provide the advantages of ease of production and lower antibody titers necessary to achieve a detectable or therapeutically effective level of antibody-target complex formation.
  • monoclonal antibodies which provide the advantages of ease of production and lower antibody titers necessary to achieve a detectable or therapeutically effective level of antibody-target complex formation.
  • bi-functional antibodies and panels of antibodies are preferred which are designed or selected to have multiple specificities for a plurality of targets, wherein at least one of these targets is an I-mf protein or I-mf analog.
  • the general methods and compositions disclosed herein for detecting and/or quantifying levels or activity of I-mf, I-mf analogs and other subject proteins and peptides of the invention provides the bases for a variety of methods to diagnose and treat aberrant myoger ⁇ c conditions and processes associated with abnormal development and disease.
  • these methods and compositions provide powerful tools to screen for agonists and antagonists of specific I-mf activities involved in such processes.
  • a particularly useful set of tools in this context includes the various antibodies provided within the invention for detecting I-mf expression, activity and localization. These antibodies can be used in a wide variety of screening and diagnostic methods, as will be apparent to the ordinarily skilled artisan.
  • polynucleotide molecules, proteins, peptides and fusion proteins of the present invention are also useful in diagnostic and therapeutic methods and to screen for compounds capable of modulating the activity or expression of I-mf and related compounds.
  • test compounds may be assessed for their ability to increase or decrease I-mf activity or expression, which assays exploit the binding of I-mf to different binding partners, such as MyoD family transcription factors.
  • animals such as mice, and cell lines may be constructed that are heterozygous or homozygous for deletions of the I-mf genes.
  • knock-out animals and cell lines may be useful as disease models and as test systems for therapeutics capable of overcoming the I-mf deletion.
  • I-mf is deleted in knock-out mice.
  • kits and multicontainer units comprising reagents and components for practicing the assay, screening, diagnostic and treatment methods of the invention.
  • kits may include, for example, probes for detecting and/or quantifying I-mf, I-mf binding partners, I-mf transcripts and the like.
  • the overlapping genomic clones 11 and 12 are depicted by shaded lines.
  • a partial restriction map of the 31.9 kb genomic region covered by the clone is shown (RI refers to the restriction endonuclease Eco RI, H3 refers to the restriction endonuclease Hind HI). Exons are depicted as striped (Exon I) or black (Exons 2-5) boxes.
  • the invention provides a novel vertebrate gene, Inhibitor of MyoD family (I-mf), and a number of corresponding polynucleotides encoding native myogenic regulatory proteins, including I-mfa, I-mfb and I-mfc. These I-mf-encoding polynucleotides and their protein products exhibit novel structural, biochemical and cell biological properties rendering them particularly useful in the study and/or control of a variety of myogenic regulatory processes.
  • I-mf Inhibitor of MyoD family
  • I-mf analogs drugs and other agents useful for evaluating and/or controlling myogenic processes in normal and abnormal vertebrate development and disease conditions, as well as for identifying, isolating and characterizing as yet unknown endogenous regulatory factors involved in myogenesis.
  • purified and isolated polynucleotides are provided that encode a native I-mf protein.
  • these polynucleotides include three, full- length I-mfcDNA clones designated I-mfa, I-mfb and I-mfc.
  • Other polynucleotides encoding native, vertebrate I-mf proteins are also provided that have variations in amino acid sequence compared to the exemplary cDNA clones described herein, such as variations among interspecific homologs and intraspecific allelic variants of the I- f gene.
  • 'hative I-mf-encoding polynucleotide refers to a polynucleotide derived from a natural source, such as a genomic or cDNA library, that hybridizes under moderate to high stringency conditions to one or more of the aforementioned full length murine cDNAs (SEQ ID NOS:l, 3 and 5).
  • 'hative I-mf-encoding polynucleotides encode products having at least 80% amino acid identity with one or more native murine I-mf proteins encoded by (SEQ ID NOS:l, 3 and 5), namely I-mfa, I-mfb and I-mfc (SEQ ID NOS:2, 4 and 6, respectively), or with a corresponding functional domain or peptide fragment of I-mfa, I-mfb and I-mfc, and exhibit one or more biological activities characteristic of one of these native I-mf proteins. Alignment of amino acid sequences and calculation of percent identity between the aligned sequences is routine in the art.
  • routine alignments include the introduction of gaps and employ other widely known conventions to account for sequence additions, deletions conservative substitutions, etc.
  • conventional sequence comparison methods involve alignment of the compared sequences to yield the highest possible alignment score, which is readily calculated based on the number of amino acid or nucleotide matches. Once optimal alignment is achieved, the degree of homology between the two sequences is determined by the formula:
  • Hybridization stringency can be altered by, for example, adjusting the temperature of hybridization, adjusting the percentage of helix-destabilizing agents such as formamide in the hybridization mix, and adjusting the temperature and salt concentration of the wash solutions.
  • the stringency of hybridization is adjusted during the post- hybridization washes by varying the salt concentration and/or the temperature. Stringency of hybridization may be reduced by reducing the percentage of formamide in the hybridization solution or by decreasing the temperature of the wash solution.
  • high stringency conditions involve, for example, high temperature hybridization (e.g., 65-68°C in aqueous solution containing 4-6X SSC, or 42°C in 50% formamide) combined with a high temperature (e.g., 5-25°C below the T m ) wash and a low salt concentration (e.g., 0.1X SSC).
  • Moderate stringency conditions involve, for example, hybridization at a temperature between 50°C and 55°C and washes in O.lx SSC, 0.1% SDS at between 50°C and 55°C, which should be sufficient to identify polynucleotide molecules encoding I-mf from other species or to isolate isoforms of I-mf.
  • low stringency conditions may involve lower hybridization temperatures (e.g., 35-42°C in 20-50% formamide) and intermediate temperature (e.g., 40-60°C) washes in a higher salt concentration (e.g., 2-6X SSC).
  • native I-mf' refers to a function or set of activities performed by a native I-mf protein in vivo or in vitro, including, but not limited to anti-myogenic activities of binding to a myogenic factor (e.g. a MyoD family transcription factor), inhibiting nuclear localization of a myogenic factor, inhibiting DNA binding of a myogenic factor, inhibiting transcriptional activation of muscle specific genes by a myogenic factor, and inhibiting myogenic differentiation of muscle phenotypes (e.g. expression of myosin heavy chain or myotube formation) in vertebrate cells, as well as non-myogenically related activities such as binding to antibodies, etc.
  • a myogenic factor e.g. a MyoD family transcription factor
  • myogenic factor e.g. a MyoD family transcription factor
  • myogenic factor e.g. a MyoD family transcription factor
  • myogenic factor e.g. a MyoD family transcription factor
  • myogenic factor e.g. a MyoD family
  • I-mf encoding polynucleotides encode three native I-mf proteins which share a common amino-terminal region. These same native I-mf proteins have distinct carboxyl termini. Exemplary proteins include the I-mf proteins shown in SEQ ID NOS:2, 4 and 6.
  • I-mf proteins These and other common and unique structural characteristics of the different native I-mf proteins disclosed herein render them particularly useful within the invention for identifying and characterizing allelic variants and interspecific homologs otl-mf for identifying and characterizing as yet unknown rriyogenic factors that interact with I-mf, and for guiding successful efforts to generate I-mf analog-encoding polynucleotides to obtain useful I-mf protein analogs for these pu ⁇ oses, as is set forth in more detail below.
  • I-mf analog-encoding polynucleotides that encode I-mf analogs.
  • 'I-mf analog- encoding polynucleotide refers to a genetically engineered, synthetic or otherwise artificially derived polynucleotide that hybridizes under moderate to high stringency conditions with one or more of the aforementioned native murine cDNAs (SEQ ID NOS:l, 3 and 5) encoding either native murine I-mfa, I-mfb or I-mfc (SEQ ID NOS:2, 4 and 6).
  • 'I-mf analog-encoding polynucleotides encode I-mf analogs having at least 80% amino acid identity with one or more of the native murine I-mf proteins I-mfa, I-mfb and I- mfc (SEQ ID NOS:2, 4 and 6), or with a corresponding functional domain or peptide fragment thereof, and exhibiting one or more detectable biological activities characteristic of native murine I-mfa, I-mfb or I-mfc, or of a corresponding functional domain or peptide fragment thereof.
  • I-mf analogs include mutant I-mf proteins generated by recombinant or synthetic methods, peptide fragments of I-mf, fusion proteins and the like inco ⁇ orating only a portion of a native I-mf protein, and up to a full length, native I-mf protein.
  • I-mf analog-encoding polynucleotides of the invention encode I-mf analogs possessing normally functional or hyperfunctional _ biological activity compared to a corresponding native I-mf protein, functional domain or peptide fragment.
  • Alternative preferred embodiments within this aspect of the invention include mutant polynucleotides that encode hypofunctional or null analogs of I-mf proteins, or of selected functional domains of I-mf such as the I-mfa interactional domain.
  • I-mfa analog-encoding polynucleotides are provided that encode I-mf analogs having null or hypofunctional anti- myogenic activity compared to anti-myogenic activity observed for corresponding native I- mfa in in-vitro and in-vivo assays, which mutants are characterized by deletions of an I-mfa carboxyl terminal interactional domain ('I-mfa interactional domain'), necessary and sufficient to mediate interactions with MyoD family of myogenic factors.
  • 'I-mfa interactional domain' an I-mfa carboxyl terminal interactional domain
  • Preferred methods to isolate and purify the polynucleotides of the invention encoding native I-mf and I-mf analogs employ a yeast two-hybrid screen to identify cDNAs encoding proteins capable of interacting with the MyoD family of proteins. Briefly, these screens are conducted generally according to the methods described by Fields and Song (Nature 340: 245, 1989; and US Patent No. 5,283,173, each of which are inco ⁇ orated by reference herein in their entirety and modified as described herein).
  • a "bait" construct is designed comprising a polynucleotide expression construct inco ⁇ orating an expression vector operably coupled with a polynucleotide encoding a LexA-MyoD fusion protein.
  • a murine embryonic cDNA fusion library containing random-primed mouse embryonic cDNA joined in-frame to a sequence encoding the VP16 activation domain is prepared as described by Hollenberg et al. (Mol. Cell. Biol. 15: 3813-3822, 1995; which is inco ⁇ orated by reference herein in its entirety).
  • the two-hybrid screen is further executed as described by Vojtek et al., Cell l ⁇ l 205-214, 1993; and Hollenberg et al., Mol. Cell. Biol. 15: 3813-3822, 1995 (each inco ⁇ orated by reference herein in its entirety and modified as described herein).
  • the yeast host strain containing multimerized LexA binding sites cloned upstream of two reporter genes, the HIS3 gene and the ⁇ -galactosidase gene each integrated into the genome is transformed with the LexA-MyoD bait construct and subsequently transformed with the fusion library. Transformants are selected and further analyzed to eliminate false positive clones and clones encoding members of the Id and E protein families, and the remaining VP16 fusions of 18 clones are identified and characterized further. To obtain the full length I-mf cDNAs a mouse embryonic library was screened using an I-mf cDNA fragment obtained from the two-hybrid screen as a probe.
  • I-mfa, I-mfb or I- mfc were subcloned and sequenced using conventional methods and reagents.
  • DNA sequencing and genomic mapping revealed that the I * -mfa, I-mfb and I-mfc transcripts are generated through differential poly-(A) adenylation and alternative splicing.
  • the genomic organization of I-mf was analyzed from two overlapping genomic clones 11 and 12 covering 31.9 kb genomic sequences. Mapping demonstrated that the I-mf gene contains five exons, and that I-mfa, I-mfb and I-mfc are alternative splicing products of I-mf.
  • conventional techniques including, for example, standard screening, subcloning and polymerase chain reactions according to the methods and using the reagents described in Sambrook et al., ibid.
  • Additional methods to isolate and purify polynucleotides of the invention encoding native I-mf and I-mf analogs utilize a variety of genomic or cDNA libraries known in the art.
  • the polynucleotide sequences encoding native I-mf and I-mf analogs may be isolated from suitable vertebrate hosts including primate, ovine, caprine, bovine, canine, feline, avian, and the like.
  • the techniques for isolating such polynucleotide sequences using probe-based methods are conventional (e.g., standard hybridization techniques are described, for example by, Sambrook et al.
  • polynucleotide expression constructs encoding an I-mf protein or I-mf analog are provided.
  • the polynucleotide expression constructs of the invention are introduced into suitable host cells to permit the expression of an I-mf protein or I-mf analog.
  • These expression constructs include a polynucleotide expression vector operably coupled with a polynucleotide encoding an I-mf protein or I-mf analog to form an expression construct operable to direct expression of the I-mf protein or I-mf functional analog by the host cell following introduction of the expression construct into the cell.
  • Exemplary I-mf based polynucleotide expression constructs of the invention inco ⁇ orate one of the three aforementioned, full-length I-mfcONA clones encoding native I-mfa, I-mfb or I-mfc. Additional examples inco ⁇ orate I-mf analog-encoding polynucleotides that hybridize under moderate to high stringency conditions to one or more of the aforementioned cDNAs encoding native I-mfa, I-mfb or I-mfc.
  • Suitable vectors for use within the polynucleotide expression constructs of the invention include DNA plasmids, DNA viral expression vectors, RNA viral expression vectors and the like.
  • the expression vector includes one or more elements that facilitate or control expression of I-mf protein or I-mf functional analog, such as a transcriptional promoter and/or enhancer sequences, a sequence encoding a retroviral transacting factor or the like. It may be preferable to include suitable signal sequences to permit the secretion of the proteins and polypeptides of the invention. Additional vector sequences include heterologous reporter sequences and sequences encoding selectable markers. The selection of suitable promoters, enhancers, terminators and the like will be determined by the selected host cell and will be evident to one skilled in the art. A wide variety of suitable yeast vectors are known (see for example, Struhl et al., Proc. Natl. Acad. Sci.
  • Such vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.
  • selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include LELJ2_(Broach et al.
  • CAT chloramphenicol resistance on yeast cells.
  • methods are provided for expressing an I- mf protein or I-mf analog in a eukaryotic host cell.
  • a suitable host cell is selected, and a polynucleotide expression construct inco ⁇ orating an I-mf based polynucleotide encoding an I-mf protein or I-mf analog is introduced into the host cell.
  • the host cell transformed or transfected with the expression construct is incubated in a suitable medium under physiological conditions to promote expression of the I-mf protein or I-mf analog by the host cell.
  • the I-mf protein or I-mf analog is isolated from the host cell or conditioned medium after its expression by the cell.
  • I-mf based expression constructs are employed in methods for modulating (i.e. enhancing or inhibiting) expression or activity of endogenous MyoD family of myogenic proteins or I- mf proteins in host cells transformed or transfected with the construct, for example to induce ectopic expression of I-mfa or an I-mfa analog and thereby inhibit a myogenic activity of a MyoD family gene or its expression product.
  • Suitable host cells for use within these methods include a variety of known and readily available fungal, bacterial and mammalian cells routinely selectable to express the I- mf proteins and I-mf analogs of the invention.
  • yeast strains of the yeast Saccharomyces cerevisiae are preferred.
  • Preferred expression vectors for use in yeast include pEMBLye30/2 (Banroques et al., Cell 46: 837-844, 1986, inco ⁇ orated herein by reference in its entirety).
  • Preferred promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl.
  • I-mf proteins and I-mf analogs of the present invention can also be expressed in filamentous fungi, for example, strains of the fungi Aspergillus (McKnight and Upshall, U.S. Patent 4,935,349, which is inco ⁇ orated herein by reference).
  • useful promoters in these cells include those derived from Aspergillus nidulans glycolytic genes, such as the ADH3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1985) and the tpiA promoter.
  • An example of a suitable terminator is the ADH3 terminator (McKnight et al., ibid.).
  • the expression constructs utilizing such components are cloned into vectors that are capable of insertion into the chromosomal DNA of Aspergillus.
  • cultured mammalian cells may be used as host cells within the present invention.
  • Preferred cell lines include NIH3T3 cells, and C3H10T1/2 cells (C3H10T1/2; Accession No. CCL 226 American Type Culture Collection (ATCC), 12301 Parklawn Drive, RockviUe, MD).
  • rodent cell lines including p3X63Ag8 (ATCC TIB 9), FO (ATCC CRL 1646), NS-1 (ATCC TIB 18) and 210-RCY- Agl (Galfre et al., Nature 277: 131, 1979); COS-1 (ATCC CRL 1650); BHK, p363.Ag.8.653 (ATCC CRL 1580) Rat Hep I (ATCC CRL 1600), Rat Hep ⁇ (ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC CCL 75.1), Human hepatoma (ATCC HTB-52), 20 Hep G2 (ATCC HB 8065), Mouse liver (ATCC CC 29.1), 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci USA 77:
  • MammaUan expression vectors for use in carrying out the present invention will include a promoter capable of directing the transcription of a cloned gene or cDNA.
  • Preferred expression vectors include pEMSVscribe (Davis et al., CeU 51: 987-1000, 1987, inco ⁇ orated herein by reference in its entirety) and pCS2 (Rupp et al., Genes Dev. 8: 1311- 1323, 1994; Turner and Weintraub, Genes Dev. g: 1434-1447, 1994, each inco ⁇ orated herein by reference in its entirety).
  • Preferred promoters include viral promoters and ceUular promoters.
  • Preferred viral promoters include the major late promoter from adenovirus 2 (Kaufman and Sha ⁇ , Mol. Cell. Biol. 2_: 1304-13199, 1982, inco ⁇ orated herein by reference in its entirety), the cytomegalo virus promoter (Boshart et al., £eH 41: 521-530, 1985, inco ⁇ orated herein by reference in its entirety) and the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1: 854-864, 1981, inco ⁇ orated herein by reference in its entirety).
  • Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., Science 222: 809-814, 1983, inco ⁇ orated herein by reference in its entirety). Also contained in the expression vectors is a transcription termination signal located downstream of the coding sequence of interest. Suitable transcription termination signals include the early or late polyadenylation signals from SV40 (Kaufman and Sha ⁇ , Mol. Cell. Biol. 2:1304-1319, 1982, inco ⁇ orated herein by reference in its entirety), the polyadenylation signal from the Adenovirus 5 elB region and the human growth hormone gene terminator (DeNoto et al., Nucleic Acid, Res.
  • Expression vectors for use within the invention may also contain a set of RNA spUce sites located downstream from the promoter and upstream from the polynucleotide sequence encoding the peptide or protein of interest.
  • Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobuUn genes.
  • the expression vectors may also include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites, as well as enhancer sequences, such as the SV40 enhancer and the mouse mu enhancer (Gillies, Cell 3_3: 717-728, 1983, inco ⁇ orated herein by reference in its entirety).
  • a noncoding viral leader sequence such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites
  • enhancer sequences such as the SV40 enhancer and the mouse mu enhancer (Gillies, Cell 3_3: 717-728, 1983, inco ⁇ orated herein by reference in its entirety).
  • Mammalian cells can also be transduced with virus such as SV40, CMV and the like.
  • virus such as SV40, CMV and the like.
  • viral vectors cloned polynucleotide molecules may be introduced by infection of susceptible ceUs with viral particles.
  • Retroviral vectors may be preferred for use in expressing I- f in mammalian cells particularly if I- f is used for gene therapy (for review, see MiUer et al. Methods in Enzvmology 217: 581-599, 1994, inco ⁇ orated herein by reference in its entirety).
  • Cloned polynucleotide sequences may be introduced into cultured mammaUan cells by a number of methods including calcium phosphate precipitation (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973; inco ⁇ orated herein by reference in its entirety); lipofection, microinjection and electroporation (Neumann et al., EMBO J. 1: 841-845, 1982; inco ⁇ orated herein by reference in its entirety).
  • a selectable marker is generally introduced into the cells along with the polynucleotide of interest.
  • Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. Selectable markers may also complement auxotrophies in the host ceU. Yet other selectable markers provide detectable signals, such as beta-galactosidase to identify ceUs containing the cloned polynucleotide molecules. Selectable markers may be amplifiable. Such amplifiable selectable markers may be used to amplify the number of sequences integrated into the host genome.
  • a pUfiable selectable markers include the DHFR gene and the DHERr cDNA (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: 2495-2499, 1983, inco ⁇ orated herein by reference in its entirety). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, MA, inco ⁇ orated herein by reference in its entirety) and the choice of selectable markers is well within the level of ordinary skill in the art.
  • Selectable markers may be introduced into the cell on a separate plasmid at the same time as the polynucleotide of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the polynucleotide of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and
  • Host ceUs containing polynucleotide expression constructs of the present invention are grown in an appropriate or suitable medium.
  • appropriate or suitable medium means a medium containing nutrients required for the growth of cells. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.
  • the medium wiU generally select for cells containing the polynucleotide expression construct by, for example, drug selection or deficiency in an essential nutrient which are complemented by the selectable marker on the polynucleotide expression construct or co-transfected with the construct.
  • Yeast ceUs are preferably grown in a chemically defined medium, comprising a non-amino acid nitrogen source, inorganic salts, vitamins and essential amino acid supplements.
  • the pH of the medium is preferably maintained at a pH greater than 2 and less than 8, preferably at pH 6.5.
  • Methods for maintaining a stable pH include buffering and constant pH control, preferably through the addition of sodium hydroxide.
  • Preferred buffering agents include succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, MO). It may also be desirable to provide an osmotic stabilizer in the medium.
  • a preferred osmotic stabilizer is sorbitol supplemented into the medium at a concentration between 0.1 M and 1.5 M., preferably at 0.5 M or 1.0 M.
  • Cultured mammaUan ceUs are generally grown in commercially available serum-containing or serum-free media. Selection of a medium appropriate for the particular ceU line used is within the level of ordinary skill in the art.
  • I-mf proteins, I-mf analogs and other subject expression products of the invention may be isolated from host ceUs grown under conditions that allow the expression and/or secretion of I-mf proteins and I-mf analogs by the host ceUs.
  • the ceU material either whole cells in the case of a secreted protein or peptide or cell debris in the case of a intracellular protein or peptide
  • the ceU material is typically separated from the conditioned medium, and the I-mf proteins, I-mf analogs or other subject expression products are isolated using separation and purification techniques known in the art.
  • Suitable isplation techniques include separation, precipitation and fractionation using a chromatographic methods, including liquid chromatography, gradient centrifugation, gel electrophoresis, gel filtration, ion exchange chromatography and immunoaffinity chromatography, among others.
  • Methods of protein purification are well known in the art (see generally, Scopes, R, Protein Purification. Springer- Verlag, NY, 1982, which is inco ⁇ orated herein by reference) and may be applied to the purification of I-mf and other subject proteins and peptides described herein. According to these general methods, I-mf and other subject proteins and peptides are provided isolated from host cells or other biological samples, and substantially free of cellular debris, other proteins and like contaminants.
  • purified I-mfa proteins, I-mf analogs, I-mf functional domains and I-mf peptide fragments are provided in a substantially pure form of at least 50% homogeneity.
  • Preferred compositions contain I-mfa proteins and other subject proteins and peptides purified to at least about 70-80% homogeneity, and more preferably to about 95-99% or more homogeneity.
  • a particularly preferred purification method is immunoaffinity chromatography using an antibody directed against an I-mf protein or an I-mf analog (e.g. an I-mf fusion protein such as HA-I-mfa, described below).
  • the antibody is preferably immobilized or attached to a solid support, substrate or other medium, for example Protein A agarose.
  • Conditioned medium or a sample from the host cells, such as a cell lysate is then contacted with the bound antibody under conditions that allow formation of complexes between the antibody and I-mf proteins, I-mf analogs or other subject expression products.
  • the complexes may be washed to remove unbound material, and the I-mf proteins, I-mf analogs or other subject expression products are released or eluted through the use of conditions unfavorable to complex formation.
  • Particularly useful methods of elution include altering pH (wherein the immobilized antibody has a high affinity for the Ugand at a first pH and a reduced affinity at a second, higher or lower, pH); changes in concentration of certain chaotropic agents; or through the use of detergents.
  • substantiaUy pure (i.e. 70-80% or greater homogeneity) native I-mf proteins including fuH length I-mfa, I-mfb and I-mfc.
  • substantially pure I-mf analogs including mutant I-mf proteins generated by recombinant or synthetic methods, functional domains and peptide fragments derived from I-mf, fusion proteins and the Uke inco ⁇ orating only a portion of a native I-mf protein and up to a full length, native I-mf protein.
  • I-mf proteins SEQ ID NOS:2, 4 and 6 which share a common amino- terminal region but have distinct carboxyl termini.
  • the carboxyl termini of I-mfa and I-mfb are approximately equal in size, and comprise approximately one-third of the full length polypeptide.
  • I-mfc SEQ ID NO: 6
  • I-mfc has a shorter unique carboxyl terminus.
  • I-mf analogs are provided which are genetically engineered, synthetic or otherwise artificially derived proteins or peptides having at least 80% amino acid identity with one or more of the native murine I-mf proteins I-mfa, I-mfb and I-mfc (SEQ ID NOS:2, 4 and 6, respectively), or with a corresponding functional domain or peptide fragment thereof.
  • GeneraUy determination of sequence identity values between I-mf analogs and their native I-mf counte ⁇ arts will follow the conservative alignment and computational principles set forth above.
  • I-mf analogs having internal deletions are aligned with a native I-mf counte ⁇ art with gaps introduced into the longer sequence and counted in a denominator of the computational algorithm to effectively reduce the derived value of percent sequence identity for the analog.
  • any non-overlapping terminal sequences or introduced sequences are counted in the longer sequence and are included in the conservative computation to effectively reduce resultant sequence identity values.
  • an I-mf analog represents a fragment, fusion portion or other partial sequence derived or engineered to only include a fraction of a corresponding native I-mf (e.g.
  • a highly truncated peptide analog of a parent peptide, or a fusion protein analog inco ⁇ orating the I-mfa C-terminal interactional domain fused with a heterologous sequence only those portions of the analog that 'correspond" to the native I- mf on a gross structural level are included in determining the length of sequences for computational pu ⁇ oses. Accordingly, terminal portions of a truncated native I-mf Source" protein truncated in a peptide fragment, extensive internaUy deleted sequences, and heterologous sequences added in a fusion protein, for example, are not considered to correspond for the pu ⁇ ose of computing sequence identity of certain I-mf analogs.
  • the I-mf analogs of the invention exhibit one or more detectable biological activities characteristic of native I-mfa, I-mfb or I-mfc, or of a corresponding functional domain or peptide fragment thereof.
  • anti-myogenically active I-mf analogs alone or joined with a heterologous sequence or agent in a fusion protein or protein-chemical conjugate, exhibit one or more detectable anti-myogenic biological activities of a native I-mf counte ⁇ art (e.g., binding to a myogenic factor, inhibiting nuclear localization of a myogenic factor, inhibiting DNA binding of a myogenic factor, inhibiting transcriptional activation of muscle specific genes by a myogenic factor, and/or inhibiting myogenic differentiation of muscle phenotypes in mammaUan cells).
  • a native I-mf counte ⁇ art e.g., binding to a myogenic factor, inhibiting nuclear localization of a myogenic factor, inhibiting DNA binding of a myogenic factor, inhibiting transcriptional activation of muscle specific genes by a myogenic factor, and/or inhibiting myogenic differentiation of muscle phenotypes in mammaUan cells.
  • I-mf analogs of the invention possess normally functional or hyperfunctional biological activity compared to corresponding native I-mf proteins, functional domains or peptide fragments.
  • Normally functional I-mf analogs exhibit approximately the same level of biological activity (e.g., an anti-myogenic activity, half-life, binding interactions with an anti- I-mf antibody, etc.) compared to a corresponding native I-mf protein, functional domain or peptide fragment, whereas the activity of hyperfunctional I-mf analogs is detectably increased relative to its native counte ⁇ art.
  • Alternative preferred embodiments within this aspect of the invention include mutant polynucleotides that encode hypofunctional or null analogs (i.e., analogs having a detectably reduced or wholly abolished biological activity compared to a native I-mf counte ⁇ art) of I-mf proteins, or of selected functional domains of I-mf such as the I-mfa interactional domain.
  • hypofunctional or null analogs i.e., analogs having a detectably reduced or wholly abolished biological activity compared to a native I-mf counte ⁇ art
  • selected functional domains of I-mf such as the I-mfa interactional domain.
  • I-mfa analogs are provided that exhibit null or hypofunctional anti-myogenic activity compared to anti-myogenic activity observed for corresponding native I-mfa in in-vitro and/or in-vivo assays, which mutants are characterized by deletions of the I-mfa carboxyl terminal interactional domain necessary and sufficienfto mediate interactions between I-mf and MyoD famUy of myogenic factors.
  • Particularly preferred I-mf analogs comprise discrete functional domains of a native I- mf protein, for example the I-mfa interactional domain.
  • Another example of an I-mf analog comprising an I-mfa interactional domain is a sequence identified by deletion mutant analysis to encode an even smaller I-mfa peptide fragment (amino acids 163-202 of SEQ ID NO:2), which binds with Myf5 and exhibits further detectable biological activity of inhibiting Myf5-mediated transcriptional activation and myodifferentiation.
  • I-mf analog comprising an I-mf functional domain
  • a sequence determined by deletion mutant expression analysis to encode an I-mfa peptide fragment corresponding to amino acids 87-163 of SEQ ID NO:2, capable of directing I-mfa self interaction.
  • I-mf analogs of the invention will be as small as possible while still maintaining substantially all of the biological activity of a larger peptide.
  • the subject peptides have an amino acid sequence encoded by a nucleic acid which hybridizes under high stringency conditions with an oligonucleotide of 15 or more contiguous nucleotides of SEQ ID NOS:l, 3, or 5.
  • Particularly preferred I-mf analogs comprise minimal functional domains of a native I-mf protein, for example the I-mfa interactional domain.
  • Minimal functional domains consist of the smaUest amino acid sequence determined by deletion mutagenesis or Uke methods combined with functional assays, as disclosed herein, to have retain least 50% (preferably at least 55%, more preferably about 90% or greater, and most preferably a hyperfunctional activity level of greater than 100%) of normal biological activity compared to that of a full-length I-mf counte ⁇ art (e.g., as determined by a CAT reporter assay such as described in Example 4 below to determine analog activity levels compared to full length I-mf activity levels in co-transfected cells, preferably including normalization of expression levels through Western blot analyses to control for differences in stabiUty between the analog and full-length I-mf counte ⁇ art in the assay).
  • a CAT reporter assay such as described in Example 4 below
  • an I-mf analog corresponding to a minimal functional domain is a smaU I-mfa peptide fragment (amino acids 163-202 of SEQ ID NO:2) which binds with Myf5 and exhibits at least 50% of normal biological activity of full length I-mfa of inhibiting Myf5 -mediated transcriptional activation and myodifferentiation.
  • I-mf analog comprising an I-mf functional domain (as determined by the specific deletion mutant analyses described herein in Example 4) is a hyperfunctional I-mfa peptide fragment corresponding to amino acids 87-163 of SEQ ED NO:2, capable of directing I-mfa self interaction. It wiU be understood by those skilled in the art that the mutant analysis conducted to determine these minimal functional domains can be readily expanded to further narrow and characterize absolute minimal functional domains within the methods of the invention. Further refinement of the minimal functional domains described herein would require only routine extrapolation from the methods and results described herein and would be expected to yield only insubstantial differences in the nature of the result obtained.
  • I-mf polynucleotide sequences encoding the I-mfa carboxyl terminal interactional domain and other I-mf functional domains, including most preferably minimal I- mf functional domains, wiU likely yield I-mf analogs that possess either hyperfunctional or hypofunctional anti-myogenic activities compared to, for example, those of native I-mfa.
  • Such mutants can be useful by themselves in various applications, or combined with other native or mutant peptide sequences, labels or other agents for more specific pu ⁇ oses.
  • an I-mf analog having a hypermo ⁇ hic mutation in the carboxyl terminal interactional domain is predicted to exhibit enhanced anti-myogenic activity, such as increased binding to a MyoD transcriptional factor or increased inhibition of myodifferentiation is transformed or transfected into suitable ceUs to express such an analog.
  • Such hyperfunctional I-mf C-terminal interactional domain mutants wiU be particularly useful as gene therapeutic agents to induce or enhance I-mf function in cells where I-mf expression or activity impaired.
  • proteins and peptides of the invention which have I-mf activity may be modified from a native I-mf sequence as necessary to provide other additional desired activities which may not have been present in a corresponding native I-mf source protein, e.g., novel or altered binding or inhibitory activities, improved adso ⁇ tion to a soUd phase, etc.).
  • hyperfunctional I-mf interactional domain mutants can be operably joined to other peptide sequences, labels or chemical agents to achieve a particular therapeutic value.
  • I-mf analogs will provide useful tools in a variety of applications, for example to screen for agonists that replace or enhance I-mf activity, or as gene therapeutic agents capable of modulating endogenous I-mf expression or activity in ceUs exhibiting aberrant myogenesis, such as CCD affected ceUs or rhabdomyosarcoma cells.
  • I-mf proteins, I-mf analogs and other subject proteins, fusion proteins and peptides of the invention are provided according to the assay and purification techniques, recombinant DNA methodologies and biosynthetic and peptide chemical techniques disclosed herein, as well as by additional methods described elsewhere and generally known in the art.
  • Uke methods for mutage ni zing, synthesizing or deriving desired proteins and peptides are widely known and routinely practiced in the art.
  • the proteins and peptides of the invention may be subjected to routine mutational changes, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes predictably provide for certain advantages in their use.
  • conservative substitutions involve replacement of an amino acid residue with another which is biologically and/or chemically sim ⁇ ar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as Gly, Ala; Val, Be, Leu; Asp, Glu; Asn, Gin; Ser,
  • sequence of the peptide will not differ by more than about 20% from the native I-mf sequence, except where additional amino acids may be added at either terminus for the pu ⁇ ose of modifying the physical or chemical properties of the peptide for, e.g., ease of linking or coupling, and the Uke.
  • peptides of the invention can be joined in a composition or admixture.
  • the peptides in the composition can be identical or different, and together they should provide reduced, equivalent, greater or qualitatively different biological activity than either of the parent peptides alone.
  • the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984); Tarn et al., J. Am. Chem. Soc.
  • Short peptide sequences, or libraries of overlapping peptides, usuaUy from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • oligonucleotide- directed mutagenic techniques that can be routinely used to generate targeted mutations within specific regions or functional domains of I-mf .based polynucleotides to yield I-mf analogs having predictable changes in activity compared to their native I-mf counte ⁇ arts.
  • Various methods for conducting site directed mutagenesis can be optionaUy employed with equal success, for example alternative one-primer and two-primer methods generally known in the art, as described generaUy in Sambrook et al., Molecular Cloning. A Laboratory Manual, ibid.; Ausubel et al., (ed.) Current Protocols in Molecular Biolog y .
  • mutagenesis may be accomplished using PCR-mediated mutagenesis using, for example, the polymerase chain reaction-mediated site-directed mutagenesis procedure described by Stappert et al. (Nucleic Acid. Res. 20: 624, 1992; which is inco ⁇ orated herein by reference in its entirety).
  • the invention provides powerful assays and reagents for further determining I-mf activity and evaluating the functional role of specific domains and residues of I-mf proteins.
  • the principles for selecting targeted mutations based on such data and employing such tools are generally known and easily implemented, and the teachings of the present disclosure greatly narrow the scope of necessary investigation, and enhances the predictabiUty of success, for conducting targeted mutational exercises within the invention.
  • I-mf analogs useful within the invention possess at least 80% amino acid identity with one or more of the native murine I-mf proteins I-mfa, I- mfb and I-mfc (SEQ ID NOS:2, 4 and 6), or with a corresponding (i.e. optimally sequence matched) functional domain or peptide fragment of I-mfa, I-mfb and or I-mfc.
  • I-mf analogs of the invention are further characterized by exhibiting one or more detectable biological activities characteristic of native murine I-mfa, I-mfb or I-mfc, or of a corresponding functional domain or peptide fragment thereof.
  • determining the presence and/or level of biological activity of an I-mf analog compared to its native I-mf counte ⁇ art is within the level of ordinary skill in the art, particularly considering the routine assay methods and powerful new assay tools provided herein for detecting I-mf, I-mf analogs, I-mf binding partners (e.g. anti-I-mf antibodies, or endogenous I-mf binding partners such as MyoD family transcription factors). Accordingly, the present disclosure provides more than sufficient direction and guidance to enable a skilled practitioner to make and use I- mf analogs having the specified structural characteristics, and to readily determine the modifications to produce such analogs Ukely to possess a predicted biological activity.
  • I-mf binding partners e.g. anti-I-mf antibodies, or endogenous I-mf binding partners such as MyoD family transcription factors
  • the subject proteins, peptides and fusion proteins of the invention are used to identify and prepare immunoreagents including antibodies, antibody derivatives, chimeric antibodies and antibody conjugates that bind specificaUy to one or more of the native I-mf proteins and I-mf analogs disclosed herein.
  • immunoreagents including antibodies, antibody derivatives, chimeric antibodies and antibody conjugates that bind specificaUy to one or more of the native I-mf proteins and I-mf analogs disclosed herein.
  • ' ⁇ specific binding is meant that the immunoreagent binds to an I-mf protein and/or I-mf analog with a detectably greater binding affinity or quantitative level of binding than a standard, non-specific control antibody (e.g., anti-keyhole Umpet hemocyanin antibody).
  • immunoreagents provided within the invention include labeled antibodies, antibody derivatives and chimeric antibodies that bind specifically to one or more of the native I-mf proteins and I-mf analogs disclosed herein, thereby providing useful probes to faciUtate detection of antibody-I-mf complexes in a sample suspected of containing I-mf.
  • the immunoreagents of the invention can be used within the methods of the invention as effective tools for detecting and/or quantifying the expression, localization and/or activity of I-mf proteins and I-mf analogs, and can be inco ⁇ orated in a wide variety of assays and screening methods disclosed herein.
  • anti-I-mf immunoreagents can themselves be used as agonists or antagonists of I-mf activity, for example by interfering with binding between I-mf and a myogenic factor such as MyoD, Myf5 or myogenin and thereby preventing or reducing anti-myogenic activity of I-mf in the presence of the antibody.
  • anti-I-mf antibody conjugates can be used as targeting agents for delivery of compounds of therapeutic interest.
  • Particularly preferred immunoreagents provided and used within the invention include monoclonal antibodies, which provide the advantages of ease of production and lower antibody titers necessary to achieve a detectable or therapeutically effective level of antibody- target complex formation.
  • bi-functional antibodies and panels of antibodies are preferred which are designed or selected to have multiple specificities for a plurality of targets, wherein at least one of these targets is an I-mf protein or I-mf analog.
  • non-human antisera or monoclonal antibodies e.g., murine, lagomo ⁇ ha, porcine, equine
  • I-mf protein or I-mf based peptides I-mf protein or I-mf based peptides.
  • antibody producing ceUs are obtained from immunized animals, immortalized and screened, or screened first for the production of an antibody that binds to the I-mf protein or peptides and then immortalized.
  • a human monoclonal antibody or portions thereof may be identified by first screening a human B-cell cDNA library for DNA molecules that encode antibodies that specifically bind to I-mf according to the method generaUy set forth by Huse et al. (Science 246: 1275-1281, 1989, inco ⁇ orated by reference herein in its entirety). The DNA molecule may then be cloned and amplified to obtain sequences that encode the antibody (or binding domain) of the desired specificity.
  • Anti-I-mf antibodies are particularly useful within the invention as labeled reagents to detect, image and/or quantify the presence or activity of I-mf, I-mf analog, or agonists or antagonists of I-mf activity in biological samples.
  • I-mf proteins or I-mf analogs are identified and/or quantified by using a labeled antibody probe, preferably monoclonal antibodies, which are contacted with biological samples, such as tissues, cells or extracts of cells, under conditions that permit formation antibody-I-mf complexes in the presence of I-mf or I-mf analog (e.g., as determined prior to or contemporaneously with the assay using a control sample having a known presence or level of I-mf).
  • unlabeled primary antibody can be used in combination with labels that are reactive with the primary antibody to detect the I-mf protein of I-mf analog.
  • the primary antibody may be detected indirectly by a labeled secondary antibody selected to bind the primary antibody.
  • labels such as chen ⁇ luminescers, particles (e.g., gold, ferritin, paramagnetic particles), fluorophores, radionuclides, enzymes, enzyme substrates, enzyme inhibitors, ligands (particularly haptens), and the like to facilitate detection and/or quantitation of I-mf.
  • particles e.g., gold, ferritin, paramagnetic particles
  • fluorophores e.g., fluorophores, radionuclides
  • enzymes e.g., enzyme substrates, enzyme inhibitors, ligands (particularly haptens), and the like to facilitate detection and/or quantitation of I-mf.
  • Immunoassays suitable for use in the present invention include, but are not Umited to, enzyme-linked immunosorbant assays, immunoblots, inhibition or competition reactions, sandwich assays, radioimmunoprecipitation, and the like, as generally described in, e.g., U.S. Pat. Nos. 4,642,285; 4,376,110; 4,016,043; 3,879,262; 3,852,157; 3,850,752; 3,839,153; 3,791,932; and Harlow and Lane, Antibodies. A Laboratory Manual. Cold Spring Harbor PubUcations, NY, 1988, each inco ⁇ orated by reference herein.
  • anti-I-mf antibodies may be used as targeting agents for the deUvery of compounds of therapeutic interest.
  • compounds of therapeutic interest include, but are not Umited to, toxins, cytostatic compounds, or proenzymes whose potential function is to activate endogenous proenzymes, to activate proenzymes from exogenous sources, or to activate enzyme cleavage sites on prodrugs.
  • bifunctional antibodies having independent antigen binding sites on each immunoglobulin molecule (as disclosed for example in Thro b. Res. Suppl, X: 83, 1990, and in The Second Annual IBC International Conference on Antibody Engineering, A. George ed., Dec. 16-18, 1991), as weU as panels of antibodies having differing specificities.
  • Bifunctional antibodies and antibody panels of particular use within the invention include antibodies and panels of antibodies that bind to both I-mf, or to multiple functional domains of the I-mf protein.
  • the subject proteins and peptides of the invention are also useful as reagents to detect, image and or quantify the presence or activity of I-mf, or of agonists or antagonists of I-mf activity, in addition to their usefulness in the preparation of anti-I-mf and anti-I-mf antibodies.
  • a coUection of I-mf proteins substantially purified are provided, including all of the I-mf isoforms disclosed herein.
  • the presence, quantity and/or activity of I-mf, I-mf analogs and other subject compounds (e.g. MyoD regulatory factors) of the invention can be determined using a variety of alternative methods and reagents, including for example Ugand binding assays using immobilized I-mf proteins or fusion proteins.
  • PCR is used to generate polynucleotide fragments encoding amino acids corresponding to a selected or putative I-mf functional domain, for example amino acids 162-202 of I-mfa (SEQ ID NO: 2) containing the I-mfa C-terminal interactional domain, or amino acids 87-163 of I-mfa containing a self- interacting functional domain of I-mf.
  • the polynucleotide fragments are then cloned into a suitable expression vector, and fusion proteins are prepared as disclosed below.
  • the fusion proteins can be endogenously labeled, for example using a covalent radiolabel or other conventional marker, or alternately labeled using antibodies that bind to the fusion protein.
  • a maltose binding protein (MBP)-I-mf fusion protein is employed (see Example 3, below) and immobilized on a soUd phase substrate.
  • the bound fusion protein was then contacted with a biological sample containing labeled MyoD proteins to detect complex formation between the bound fusion protein and MyoD probe.
  • I- mf fusion protein-MyoD complexes were detected using SDS polyacrylamide gel chromatography and autoradiography.
  • Ugand overlay assays using peptides derived from MyoD family members can further refine the accuracy and results of such assays, for example to closely map I-mf interactional domains or interactional domains of I-mf binding partners, including MyoD transcription factors.
  • I-mf, I-mf analogs or other subject molecules of the invention are immobUized or attached to a substrate or support material, such as polymeric tubes, beads, polysaccharide particulates, polysacch aride-containing materials, polyacrylamide or other water insoluble polymeric materials.
  • substrate or support material such as polymeric tubes, beads, polysaccharide particulates, polysacch aride-containing materials, polyacrylamide or other water insoluble polymeric materials.
  • CNBr activation A common method of immobilization is CNBr activation.
  • Activated substrates are commerciaUy available from a number of suppUers, including Pharmacia (Piscataway, NJ), Pierce Chemical Co. (Rockford, IL) fmd Bio-Rad Laboratories (Richmond, CA).
  • a preferred substrate is CNBr -activated Sepharose (Pharmacia, Piscataway, NJ).
  • a substrate I-mf complex will be in the form of a column, and a biological sample, such as a cell or tissue extract, is contacted with the column under conditions that allow binding to occur between I-mf and any I-mf binding partners present in the sample.
  • the substrate with immobilized I-mf is first equilibrated with a buffer solution of a composition in which the I-mf has been previously found to bind its Ugand.
  • the sample in the solution used for equiUbration, is then appUed to the substrate/I-mf complex.
  • the sample be passed over the column two or more times to permit fuU binding of I-mf binding partner to the substrate/I-mf complex.
  • the substrate/I-mf complex is then washed with the same solution to elute unbound material.
  • a second wash solution may be used to minimize nonspecific binding.
  • the bound material may then be released or eluted through the use of conditions unfavorable to complex formation.
  • Particularly useful methods include changes in pH, wherein the immobilized I-mf has a high affinity for the I-mf binding partner at a first pH and a reduced affinity at a second (higher or lower) pH; changes in concentration of certain chaotropic agents; or application of detergents.
  • I-mf and I-mf analogs can be further evaluated and inco ⁇ orated within the methods of the invention with the aid of protein crystallography.
  • the basic protein expression methods of the invention provide for the production of sufficiently large amounts of pure I-mf protein to allow crystaUographic analysis of I-mf bound and unbound to their various binding partners. These studies wiU further elucidate the complex structure and function of the I-mf proteins and will be of substantial value in the design of sma molecule inhibitors of these proteins.
  • assay, screening, diagnostic and/or therapeutic methods and compositions are provided that employ one or more of the aforementioned inventive tools.
  • probes corresponding to I-mf proteins, I-mf analogs, anti-I-mf antibodies and the like are used to detect and or quantify expression and/or activity of I-mf in biological samples.
  • a biological sample e.g. a mammalian cell lysate
  • the sample is contacted with an I-mf specific probe (e.g.
  • an antibody that specificaUy binds to I-mf protein or I-mf analog, or another I-mf binding partner that detectably binds with I-mf, such as a MyoD family myogenic factor) under conditions that permit formation of detectable complexes between the probe and I-mf protein or I-mf analog in a suitable control sample e.g. a control sample having a known amount of I-mf and contacted with the probe under the same conditions as the biological sample to demonstrate, and/or provide a standard value for quantifying, formation of detectable complexes.
  • a suitable control sample e.g. a control sample having a known amount of I-mf and contacted with the probe under the same conditions as the biological sample to demonstrate, and/or provide a standard value for quantifying, formation of detectable complexes.
  • labeled I-mf or I-mf analogs are themselves used as probes to detect and or quantify binding between the I-mf probe and a known or unknown I-mf binding partner, such as a MyoD family myogenic factor.
  • a biological sample is contacted with an I-mf or I-mf analog probe under conditions that permit formation of detectable complexes between the probe and an I-mf binding partner in a suitable control sample containing the binding partner.
  • the presence and/or amount of complexes formed between the I-mf probe and I-mf binding partner is then determined in the biological sample (e.g.
  • I-mf binding partner is present in the biological sample and, if desired, at what level. In this manner, unknown I-mf binding partners can also be detected and, if present, purified and isolated to determine its structure and activity.
  • the basic assay methods set forth above allow detection and quantification of I-mf and I-mf binding partners to assess the expression, location and activity of these factors during normal and abnormal development and myogenic differentiation. As such, these methods can be directly inco ⁇ orated into methods to screen and diagnose abnormal developmental and disease conditions in mammals involving defective expression or activity of I-mf or one of its binding partners.
  • diagnostic samples are provided from a patient at risk for a developmental defect or disease involving aberrant expression or activity of I-mf or an I-mf binding partner, and the sample is assayed as above to detect and/or quantify I-mf or I-mf binding partner in the sample.
  • Increased or decreased expression or activity of I-mf or I-mf binding partner in the biological sample (e.g. relative to a level of expression or activity established as a normal baseUne in control patient samples) can then be determined as an indicator of the occurrence or risk of related developmental defects or diseases in the patient being diagnosed.
  • the foregoing assay methods provide the basis for a variety of methods to screen for modulators of I-mf or I-mf binding partner expression or activity.
  • labeled I-mf proteins or I- mf analogs, anti-I-mf antibodies and the like are used as reagents to screen small molecule and peptide libraries to identify inhibitors of I-mf gene expression or I-mf protein activity.
  • I-mf proteins or I-mf analogs are used as competitive probes (either labeled directly, e.g. covalently, or secondarily, e.g.
  • I-mf binding partners such as an anti-I-mf antibody or a MyoD family myogenic factor, is used as a probe.
  • a test sample e.g. a mammalian cell lysate to which a test substance has been added
  • an I-mf specific probe e.g.
  • an antibody that specifically binds to I-mf protein or an I-mf binding partner that detectably binds I-mf such as a MyoD family myogenic factor under conditions that permit formation of detectable complexes between the probe and I-mf protein in the absence of the test substance (e.g. in a control sample having a known amount of I-mf to which no test substance has been added, which is also contacted with the probe under the same conditions as the test sample to demonstrate, and or provide, prior to the assay or contemporaneously therewith, a standard value for detecting and/or quantifying formation of complexes).
  • test substances useful within the invention may include substances present throughout the handling of test sample components, for example host ceU factors that are present in a ceU lysate used for generating the test sample. Such endogenous factors may be segregated between the test and control samples for example by using different cell types for preparing lysates, where the ceU type used for preparing the test sample expresses a putative test substance that is not expressed by the ceU type used in preparing the control sample.
  • a particularly useful set of test and control ceU types in this context are ceUs that have and have not been induced to undergo myodifferentiation by transformation with a MyoD factor.
  • the presence and/or amount of complexes formed between I-mf and the I-mf specific probe is detected in the test sample (e.g. by determining the presence or amount of label bound directly to the probe or to a secondary antibody directed against the probe), indicating whether increased or decreased expression or activity of I-mf (i.e. compared to a level of expression or activity estabUshed as a relevant control value) resulted in the test sample due to the presence of the test substance.
  • More specific screening methods are also disclosed herein, including Ugand overlay assays using peptides derived from I-mf binding partners as test substances (for example according to the methods of Chakraborty et al. EMBO J. 14: 1314-1321, 1995) in assay mixtures and under suitable conditions that permit binding of I-mf to the same or different binding partner in a control sample.
  • an overlapping Ubrary of chemically synthesized peptides is generated covering selected sequences of a known I-mf binding partner, preferably a MyoD family myogenic factor, and members of this Ubrary are screened as test substances using labeled I-mf, labeled anti-I-mf antibody or Uke probes according to the above methods.
  • Such screens allow the identification of highly specific peptide inhibitors of I-mf binding activity, which can in turn provide useful reagents for mapping essential and non-essential portions of I-mf proteins necessary for binding and other biological activities.
  • oligonucleotide probes corresponding to, or complementary to, portions of the I-mf gene are used to detect and/or quantify I-mf gene expression according to the general methods outlined above.
  • the invention allows further identification and isolation of endogenous regulatory factors and exogenous substance that can modulate I-mf gene expression (e.g.
  • I-mf oUgonucleotide probes are also useful within diagnostic methods of the invention, for example to map the chromosomal location of I- f and genes that may be regulated by I-mf, to detect and characterize I- f alleles, and to identify and characterize genetic defects in I-mf genes present in cell populations or individuals.
  • assays employing I-mf protein or anti-I-mf antibodies are adaptable for use within diagnostic methods
  • assays employing I-mf oligonucleotide probes can be applied to detect and/or quantify expression of I-mf or related genes in diagnostic samples from patients to determine the presence or risk of myogenic diseases involving aberrant I-mf expression.
  • Other reagents that can be used within this context include PCR primers designed to detect the presence of mutant I-mf or aberrant expression of I-mf in diagnostic samples.
  • FIG. 1 depicts a schematic diagram of a cell transformed or transfected with expression constructs inco ⁇ orating I-mf-encoding, I-mf analog-encoding or I-mf binding partner encoding polynucleotides to provide an in vivo assay mixture.
  • suitable host cells are microinjected or otherwise exposed intracellularly to I-mf, I-mf analog or I-mf binding partner to provide an in vivo assay mixture.
  • Cells thus transformed, transfected or intracellularly exposed can be used, for example, in screens to detect endogenous factors that interact with I-mf or to identify therapeutic compounds capable of modulating I-mf function.
  • in vivo screening methods employ recombinant cell lines, ova, transgenic embryos and animals manipulated to disrupt I-mf gene expression or to express native or mutant forms of I-mf, including dominant-negative and "knock-out" recombinants in which the activity of I-mf protein is down-regulated or eliminated.
  • These subject ceU lines and animals find uses in screening for candidate therapeutic agents capable of either blocking or substituting for a function performed by I-mf or correcting a cellular defect caused by I- mf.
  • the polynucleotide molecules of the present invention may be joined to reporter genes, _
  • mice are generated by replacing the murine I-mf coding region with the neomycin resistance gene to assess the consequences of eliminating the murine I-mf protein.
  • mice are useful for example as model systems for screening compounds that may developmentally, spatiaUy and/or quantitatively alter the expression of the reporter gene. Such mice may be used to study methods to rescue homozygous mutants and as hosts to test transplant tissue for treating diseases or other conditions characterized by aberrant regulation of myogenic processes.
  • the invention provides a variety of disease diagnostic and therapeutic methods and compositions for evaluating and/or treating diseases and other conditions involving aberrant myogenic regulation.
  • One potential target in this context is the human disease cleidocranial displasia (CCD).
  • CCD occurs both as an autosomal dominant and a recessive mutation and results in the delayed ossification of bones.
  • Patients with CCD have bone deformation and in some cases some level of mental retardation.
  • I- f has been mapped to a murine chromosomal location syntenic with the human chromosomal map site 6p21, at which location mutations have been found to be associated with CCD (Nienhaus et al., Am. J. Med. Genet. 46: 630-631, 1993; Feldman et al., Am. J. Med. Genet. 56: 938-943, 1995 and Mundlos et al.. Hum. Mol. Genet. 4: 71-75, 1995).
  • This physical proximity between CCD related mutations and the I-mf map site suggests that aberrant I-mf expression and or activity may well be a factor in CCD.
  • the various methods and compositions of the invention provide a useful assemblage of tools to study and potentially treat this aberrant condition.
  • Potential tools in this regard include the I-mf-encoding polynucleotides and expression constructs, I-mf transformed cells, I-mf based proteins and antibodies, oligonucleotides, as well as antisense polynucleotides and ribozymes that specificaUy target I-mf polynucleotides.
  • Other diseases targeted for diagnosis and treatment using the methods and compositions of the invention include rhabdomyosarcomas and other cancers affecting muscle tissue in mammals.
  • I-mf may be overexpressed, or that the I-mf protein may be hyperactive, in these cells.
  • inhibitors of I-mf disclosed herein which block or impair anti-myogenic activity of I-mf may alleviate or curtail aberrant myogenic regulation in rhabdomyosarcoma cells.
  • labeled I-mf proteins, I-mf analogs, and I-mf binding partners including anti-I-mf antibodies are employed to detect expression, localization and/or activity of I-mf in the context of normal and/or abnormal myogenic processes or conditions, or in association with specific molecular factors known to be involved in such processes.
  • I-mf expression or activity is detected and/or quantified in a normal ceU population or tissue from a human source, and these results are compared to I-mf expression or activity detected and/or quantified in a test ceU population or tissue from a patient at risk for a myogenic disease (for example, a CCD patient or a patient presenting with rhabdomyosarcoma). Detection and/or quantification of
  • I-mf expression, localization or activity can be accomplished by a variety of methods, such as by in situ hybridization using anti-I-mf antibodies, by Western blotting or immunoprecipitation using anti-I-mf antibodies in contact with ceU or tissue lysates, or by affinity purification using anti-I-mf antibodies bound to a solid phase, among other methods.
  • Comparable methods are disclosed herein, or are elsewhere disclosed and known in the art, for using non-antibody agents to detect and/or quantify expression or activity of targeted compounds such as I-mf.
  • Suitable non-antibody probes for use within these methods include, for example, oUgonucleotide probes that hybridize to I-mf transcripts, labeled binding partners of I-mf, and synthetic or recombinant peptide analogs of I-mf, among other useful probe types disclosed herein.
  • I-mf cDNA and oligonucleotide probes may be useful in Northern, Southern, and dot-blot assays for identifying and quantifying the level of expression of I-mf in normal and diseased ceUs.
  • Differences that are detected and/or quantified between I-mf expression or activity between normal and test cell populations or tissues may be diagnostic of particular disease states or other conditions characterized by aberrant myogenesis, and may therefore be predictive of the risk or extent of disease, and the outcome of selected courses of treatment.
  • Preferred diagnostic methods in this context rely on labeled polynucleotide probes to map the chromosomal locations of I-mf and related genes, to determine linkage of these genes relative to other genes, and to identify genetic defects in these genes among cell populations or individuals.
  • Additional polynucleotide probes that are useful within the invention include I-mf probes corresponding to partial or complete nucleotide sequences generated from the cDNAs depicted in SEQ ID. NOS:l, 3 or 5 below, and degenerate oligonucleotides based on the amino acid sequences of SEQ ID NOS:2, 4 and 6.
  • DNA probes and PCR primers are designed as reagents for diagnostic assays for detecting the presence of I-mf or I-mf mutant sequences.
  • the nature of the specific assay may depend on the type of mutational analysis to be carried out and the type of biological sample to be assayed.
  • High molecular weight DNA may be obtained from suitable sources using commercially available kits.
  • Commercially available kits include, the Genomic Isolation Kit A.S.A.P.
  • PCR primers find used in the amplification of I-mf sequences from normal and diseased tissue. Such sequences may be analyzed by direct sequence analysis or by a variety of means to distinguish normal from mutant sequence.
  • a comparison of fragment size and or comparison of sequences may be used to diagnose a number of diseases such as cancer.
  • I-mf-specific DNA probes are used in restriction fragment length polymo ⁇ hism (RFLP) assays on DNA samples isolated from normal and diseased tissues to detect rearrangements and or deletions of the I-mf locus. More subtle mutations may be detected by a variety of methods which include but are not restricted to single strand conformation polymo ⁇ hism (SSCP) (Orita et al., Proc. Natl. Acad. Sci.
  • oligonucleotide primers may be designed to permit the amplification of sequences in the I-mf gene that may then be analyzed by either direct sequencing or other indirect methods such as SSCP to identify mutations within the I- mf gene.
  • Particularly preferred regions for designing oligonucleotide primers include intron- exon junctions. Five exons have been mapped for I-mf. Intron-exon junctions are identified when the coding sequence is interrupted. Oligonucleotides for these assays are preferably designed from flanking intron sequences.
  • the diagnostic and screening methods of the invention employing DNA probes and PCR primers find use for individuals suspected of being at risk for developing a I-mf- associated disease such as CCD (e.g., family history of disease) or for patients in which such a screening is used to diagnose or eliminate I-mf-associated disease as a causative agent behind a patient's symptoms.
  • f methods for screening use a biological samples from a patient (e.g., tissue biopsy or amniotic fluid sample) which is screened for the presence of mutations in I-mf.
  • a patient's I- f gene is compared to normal /-/n/DNA (i.e., wild-type I-mf) using a variety of methods, including RFLP analyses, SSCP, and the like, and mutations in I-mf are detected.
  • An aberrant I-mf DNA size pattern such as for RFLP analysis or SSCP analysis, aberrant I-mf protein and/or aberrant I-mf protein levels as determined by antibody assays would indicate that the patient has developed or is at risk to develop a I-mf-associated disease.
  • Prenatal diagnosis can be performed when desired, using a variety of methods to obtain fetal cells. These methods include, but are not limited to amniocentesis, chorionic villous sampling and fetoscopy. Prenatal analysis of the I-mf gene is carried out using SSCP, RFLP, DDF and the like.
  • the invention provides a range of therapeutic compositions and methods for preventing and/or treating diseases and other conditions involve aberrant myogenesis, for example CCD and rhabdomyosarcoma. Therapeutic methods of the invention variously utilize I-mf-encoding _
  • polynucleotides I-mf proteins and analogs, I-mf binding antibodies and other binding partners, expression constructs inco ⁇ orating I-mf-encoding polynucleotides, host ceUs transformed to express I-mf, and other therapeutic agents identified within the invention as modulators of I-mf expression and/or activity, including triplex forming ohgonucleotides, antisense polynucleotides and ribozymes that specifically target I-mf encoding polynucleotides.
  • agents that inhibit or enhance I-mf expression or activity In preferred therapeutic methods directed to the treatment of myogenic disease, it is useful to employ agents that inhibit or enhance I-mf expression or activity and which thereby can eliminate or impair aberrant myogenic processes.
  • Useful agents in this context generally include agents that impair or enhance I-mf binding to its partners or otherwise enhance I-mf expression or activity, for example mutant I-mf based expression constructs that induce or enhance I-mf expression or activity in targeted ceUs.
  • Therapeutic substances which can serve as inhibitors or antagonists of I-mf include, but are not limited to, compounds capable of inhibiting the formation of I-mf -MyoD family member binding complexes, compounds that reduce or inhibit the activity of I-m£ and compounds that interfere with the expression of I-mf protein.
  • Such agents may include chemical compound inhibitors of I-mf, protein, peptide or antibody I-mf antagonists, and molecules that inhibit the expression of I-mf such as triplex forming oligonucleotides, antisense polynucleotides, ribozymes, etc.
  • antisense polynucleotides and their applications are described generally in, for example, Mol and Van der Krul, eds., Antisense Nucleic Acids and Proteins Fundamentals and Applications. New York, NY, 1992, inco ⁇ orated by reference herein in its entirety.
  • Suitable antisense ohgonucleotides are at least 15 nucleotides in length and up to and including the upstream untranslated and associated coding sequences of I-mf.
  • the optimal length of antisense oUgonucleotides is dependent on the strength of the interaction between the antisense oligonucleotides and their complementary sequence on the mRNA, the temperature and ionic environment in which translation takes place, the base sequence of the antisense oligonucleotide, the presence of secondary and tertiary structure in the mRNA and/or in the antisense oUgonucleotide and the preferred delivery mode.
  • soluble antisense oligonucleotides have been used to inhibit transcription translation of a target gene (Ching et al., Proc. Natl. Acad. Sci. USA 86:10006-10010, 1989; Broder et al., Ann. Int. Med. 113: 604-618 (1990); Loreau et al., FEBS Letters 274:53-56 1990; Holcenberg et al., WO91/11535; U.S.S.N. 07/530,165 ("New human CRIPTO gene"); WO91/09865; WO91/04753; WO90/13641; and EP 386563, each inco ⁇ orated herein by reference).
  • Suitable target sequences for antisense polynucleotides include intron-exon junctions (to prevent proper splicing), regions in which DNA/RNA hybrids wiU prevent transport of mRNA from the nucleus to the cytoplasm, initiation factor binding sites, ribosome binding sites, and sites that interfere with ribosome progression.
  • a particularly preferred target region for antisense polynucleotides is the 5' untranslated region of the gene of interest, for example the I-mf gene.
  • Antisense polynucleotides targeted to the I-mf gene may also be prepared by inserting a DNA molecule containing the target polynucleotide sequence into a suitable expression vector such that the DNA molecule is inserted downstream of a promoter in a reverse orientation as compared to the gene itself.
  • the expression vector may then be transduced, transformed or transfected into a suitable cell resulting in the expression of antisense polynucleotides.
  • antisense ohgonucleotides may be synthesized using standard manual or automated synthesis techniques. Synthesized oligonucleotides may be introduced into suitable ceUs by a variety of means including electroporation (e.g., as described in Yang et al., Nucl.
  • a suitable antisense oUgonucleotide administration method wiU be evident to one skilled in the art.
  • the stabiUty of antisense oligonucleotide-mRNA hybrids may be increased by the addition of stabUizing agents to the oligonucleotide.
  • Stabilizing agents include intercalating agents that are covalently attached to either or both ends of the oUgonucleotide.
  • Oligonucleotides may be made resistant to nucleases by, for example, modifications to the phosphodiester backbone by the introduction of phosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates, phosphoramidates or phosphorodithioates. Oligonucleotides may also be made nuclease resistant by the synthesis of the oligonucleotides with alpha-anomers of the deoxyribonucleotides, as generaUy described in Mol and Van der Krul (ibid.).
  • polynucleotide-based inhibitors of the present invention include the triplex forming ohgonucleotides, sequence-specific DNA binding drugs that interfere with target gene transcription.
  • Triplex-forming oligonucleotides are generally described in Maher, Bioessays 14: 807-815, 1992; Gee et al., Gene 149: 109-114, 1994; Noonberg et al., Gene 149: 123-126, 1994; Song et al., Ann. NY Acad. Sci. 761 : 97-108, 1995; Westin et al., Nuc. Acids. Res. 23: 2184-2191, 1995; and Wand and Glazer, J. Biol. _
  • oligonucleotides form triple helical complexes under physiological conditions on double- stranded DNA, selectively inhibiting gene transcription by physically blocking RNA polymerase or transcription factor access to the DNA template. See also, e.g., WO 95/25818; WO 95/20404; WO 94/15616; WO 94/04550; and WO 93/09788, each of which is inco ⁇ orated herein by reference.
  • the triplex forming oligonucleotides may contain either a nucleotide or non-nucleotide taU to enhance the inhibition of transcription factor binding.
  • the triplex forming oligonucleotides are targeted to the I-mf gene.
  • a suitable sequence wiU be guided by, for example, the type of inhibitor (i.e., triplex forming oUgonucleotide or antisense polynucleotide) and the species to be treated. It may be preferable to choose sequences that are conserved between species to permit use in readily available animal models.
  • the present invention also provides compositions and methods for using ribozyme inhibitors for inhibiting I-mf expression.
  • the ribozymes can be administered in a variety of ways, including by gene therapy targeted to a desired cell.
  • a ribozyme of the invention is a targets the RNA transcripts of the gene of interest in a sequence-specific manner.
  • ribozymes may be designed to specifically inhibit the transcription of the I-mf gene
  • Each ribozyme molecule is designed to contain a catalytically active segment capable of cleaving 7-w/ " RNA, and further comprises flanking sequences having a nucleotide sequence complementary to portions of the targeted RNA.
  • flanking sequences serve to anneal the ribozyme to the RNA in a site-specific manner. Absolute complimentary of the flanking sequences to the target I-mf sequence is not necessary, however, as only an amount of complimentary sufficient to form a duplex with the target RNA and to aUow the catalytically active segment of the ribozyme to cleave at the target sites is necessary. Thus, only sufficient complimentary to permit the ribozyme to be hybridizable with the target RNA under physiological conditions is required.
  • ribozyme means an RNA molecule having an enzymatic activity that is able to cleave or splice other separate RNA molecules in a nucleotide base sequence specific manner.
  • catalytic or enzymatic RNA molecule is meant an RNA molecule which has complementarity in a substrate binding region to a specific RNA target (e.g. 7-w " RNA), and also has enzymatic activity that is active to cleave and/or spUce RNA in that target, thereby altering the target molecule.
  • the enzymatic RNA molecule is formed in a hammerhead motif, but the ribozyme may also be formed in the motif of a hai ⁇ in, hepatitis delta virus, group I intron or RNAse P RNA (in association with an RNA guide sequence).
  • hammerhead motifs are described by Rossi et al., AIDS Res. Hum. Retrovir. 8: 183, 1992
  • hai ⁇ in motifs are described by Hampel et al., Biochem. 28:4929, 1989 and Hampel et al., Nucl. Acids Res. 18: 299, 1990
  • the hepatitis delta virus motif is exemplified in Perrotta and Been, Biochem.
  • RNAseP motif is described in Guerrier-Takada et al., Ce 35: 849, 1983, and examples of the group I intron motif are described in Cech et al., U.S. Patent 4,987,071, each of the foregoing disclosures being inco ⁇ orated herein by reference.
  • These specific motifs are not limiting in the present invention and those of skiU in the art wiU recognize that an enzymatic RNA molecule of the invention has a specific substrate binding site which is complementary to one or more of the target RNA regions and that it has nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
  • flanking sequences upstream and downstream of the ribozyme catalytic site may comprise segments of any length that effectively imparts the desired degree of targeting specificity for the ribozyme.
  • a flanking sequence comprises from about 4 to about 24 nucleotides, more preferably from about 6 to about 15 nucleotides, and typicaUy about 9 to 12 nucleotides, and results in base pairing to the substrate sequence immediately upstream and downstream of the RNA sequences which comprise the cleavage site.
  • the I-mf modulating agents, including I-mf inhibitors and agonists, of the present invention may be used in a variety of therapeutic contexts, alone or in combination, and may be formulated for a variety of modes of administration.
  • the inhibitor or agonist may include systemic, topical or local administration. Techniques and formulations are generally described in Remington's Pharmaceutical Sciences. Mack PubUshing Co., Easton, PA, latest edition, inco ⁇ orated herein by reference in its entirety.
  • the inhibitor or agonist is generaUy combined with a pharmaceutically acceptable carrier such as a diluent or excipient. Suitable carriers may include fiUers, extenders, binders, wetting agents, disintegrants, surface-active agents or lubricants. The choice of such ingredients wiU depend on the mode of administration and dosage forms. Typical dosage forms include tablets, powders, Uquid preparation including suspensions, emulsions, and solutions, granules, capsules and suppositories. Liquid preparation for injection are also typical and include liposome preparations. __
  • Polynucleotide inhibitors e.g., triplex forming oligonucleotides, antisense oligonucleotide, ribozyme, etc., or a combination of such inhibitors targeted to different portions of the target DNA or corresponding RNA can be delivered in a wide variety of ways to targeted cells to faciUtate inhibition of the gene of interest.
  • multiple polynucleotide inhibitors of I-mf axe combined and deUvered to target cells to decrease the expression of I-mf, thus, for example, permitting an increase in the expression or activity of endogenous MyoD family proteins.
  • the oligonucleotides can be administered as synthetic oligonucleotides or expressed from an expression vector.
  • the oligonucleotide can be administered ex vivo, i.e., contacted with target cells that have been removed from an individual or other cell source, treated and returned, or the oligonucleotide molecule can be administered in vivo.
  • the target ceUs are exposed to mitogens, e.g., serum mitogens (SCF, IL-3, EPO, TPO, etc.) or the Uke depending on the particular cell population.
  • mitogens e.g., serum mitogens (SCF, IL-3, EPO, TPO, etc.
  • DeUvery of inhibitors or agonists to a targeted ceU population can be via a wide range of available deUvery vehicles, e.g., a Uposome, a controUed release vehicle, by use of iontophoresis, electroporation or ion paired molecules, or covalently attached adducts, and other pharmacologicaUy acceptable methods of delivery.
  • a carrier provides a means to accumulate the inhibitor or agonist within or at a desired ceU population.
  • the delivery vehicle can be designed to serve as a slow release reservoir or to deUver its contents directly to the target cell.
  • oligonucleotide delivery vehicles include liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and microspheres. Liposomes can readily be targeted to the various tissues or cell populations.
  • routes of administration for I-mf inhibitors and agonists include intramuscular, aerosol, intravenous, parenteral, intraperitoneal, etc.
  • the specific delivery route for a selected inhibitor or agonist will depend on a variety of factors, such as the form of the inhibitor or agonist, the intended target, the condition being treated, etc.
  • modifications can be made to enhance ceUular uptake, e.g., by reducing the oligonucleotide's charge to produce a molecule which is able to diffuse across the ceU membrane.
  • the structural requirements necessary to maintain oUgonucleotide activity and activity of other classes of inhibitors or agonists are generally recognized in the art. Modifications to enhance cellular delivery can also be designed to reduce susceptibiUty to nuclease digestion or other types of degradation.
  • the dosage of inhibitor or agonist will also depend on a variety of factors, such as the form of the inhibitor or agonist, the route of administration, etc., and thus can vary widely. Generally the dosage of inhibitors will result in inhibition of I-mf activity or levels to a sufficient extent within the targeted cells sufficient to prevent formation of I-mf complexes with its binding partners.
  • Establishment of effective levels of inhibitor within a targeted ceU population depends upon, e.g., the rate of uptake (or expression by a particular vector), and rate at which the inhibitor is degraded.
  • the duration of treatment may extend for a time sufficient to permit, e.g., transduction of a relatively high percentage of dividing ceUs compared to an untreated control ceU population, but usuaUy wiU be at least for about 2-4 days, sometimes 6-10 days, although longer durations may be necessary for quiescent or terminally differentiated ceU populations.
  • the number and timing of doses can vary considerably, depending on the factors discussed above and the efficacy of a particular inhibitor or mixture thereof, the delivery vehicle and route of administration, etc.
  • oUgonucleotide inhibitors of the present invention for example I-mf antisense oligonucleotide inhibitors or I-mf-specific triplex forming ohgonucleotides, it may be preferable to include an effective concentration of a Upid formulation with the oUgonucleotide. Suitable Upid formulations and concentrations are those that enhance the uptake of the oligonucleotides by ceUs.
  • Such lipids include cationic lipids used for lipofection such as N-[l-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) and dioleoyl phophatidylethanolamine (DOPE).
  • DOTMA N-[l-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride
  • DOPE dioleoyl phophatidylethanolamine
  • I-mf inhibitors or agonists may be used in combination with other compounds that are useful, for example in chemotherapy or as antibiotics.
  • These compounds include standard chemotherapeutic agents such as platin compounds (e.g. cisplatin) and antibiotics such as peniciUin, tetracycline or antiviral agents such as protease inhibitors and the Uke.
  • the I-mf encoding polynucleotide expression constructs disclosed herein are employed in methods for modulating the expression of I-m ⁇ disrupting I-mf expression or inducing ectopic expression of I-mf genes and related polynucleotide sequences in the context of gene therapeutic methods involving mammaUan host ceUs.
  • an I-mf analog is expressed in mammalian cells that functions to impair anti-myogenic activities of endogenous I-mf, thereby restoring myogenic differentiation under the direction of MyoD family members.
  • a suitable gene therapy vector delivery system e.g., a retroviral vector
  • a microinjection technique see, for example, Tarn, Basic Life Sciences 37: 187-194, 1986, inco ⁇ orated by reference herein in its entirety
  • a transfection method e.g., naked or liposome encapsulated DNA or RNA
  • a suitable gene therapy vector delivery system e.g., a retroviral vector
  • a microinjection technique see, for example, Tarn, Basic Life Sciences 37: 187-194, 1986, inco ⁇ orated by reference herein in its entirety
  • a transfection method e.g., naked or liposome encapsulated DNA or RNA
  • Gene transfer vectors e.g., retroviral vectors, and the like
  • Gene therapy may be used to correct conditions involving defective myogenic processes.
  • the introduction method may be chosen to achieve a transient expression of I- mf in the host ceU, or it may be preferable to achieve constitutive, tissue specific, or ind ⁇ cible expression.
  • kits and multicontainer units comprising reagents and components for practicing the assay, screening, diagnostic and therapeutic methods of the invention.
  • Exemplary kits of the invention contain reagents for detecting I- mf, I-mf-analogs, I-mf binding partners or the like, preferably included with selected additional reagents (e.g. enzymatic reagents such as reverse transcriptase or polymerase; suitable buffers; nucleoside triphosphates; suitable labels for labeling the reagents for detecting I-mf and developing reagents for detecting the signal from the label).
  • additional reagents e.g. enzymatic reagents such as reverse transcriptase or polymerase
  • suitable buffers e.g. enzymatic reagents such as reverse transcriptase or polymerase
  • nucleoside triphosphates e.g. enzymatic reagents such as reverse transcriptase or polymerase
  • kits of the invention include sequence-specific oUgonucleotide primers for detecting polynucleotide molecules encoding I-mf. Such primers may be provided in separate containers or may be provided in combinations of one or more primer pairs in a series of containers.
  • kits contain antibodies useful for detecting I-mf (or mutants thereof) in a sample. The I-mf-specific antibodies may be labeled or may be detected by binding to a secondary antibody.
  • the antibody reagents may be provided in a separate container or may be provided in combination in a series of containers.
  • the kits may also contain instructions for carrying out the assay and/or additional containers suitable for carrying out the reactions of the assay.
  • I-mf proteins, I-mf analogs, anti-I-mf antibodies and other subject molecules of the invention may also be utilized within diagnostic kits.
  • I-mf or I-mf analogs are provided in a lyophilized form or immobilized onto the walls of a suitable container, either alone or in conjunction with antibodies capable of binding to the I- mf or I-mf analog.
  • the antibodies which may be conjugated to a label or unconjugated, are generally included in the kits with suitable buffers, such as phosphate, stabilizers, inert proteins or the like.
  • kits wiU typically include other standard reagents, instructions and, depending upon the nature of the label involved, reactants that are required to produce a detectable product. Where an antibody capable of binding to I-mf is employed, this antibody wiU usually be provided in a separate vial. The antibody is typicaUy conjugated to a label and formulated in an analogous manner with the formulations briefly described above.
  • kits including the containers, may be produced and packaged using conventional kit manufacturing procedures.
  • kits including therapeutic kits, are contemplated within the invention that consoUdate materials and faciUtate performance of the methods described herein.
  • the kits may also contain instructions for carrying out the methods of the invention.
  • the following examples are offered by way of illustration, not by way of limitation.
  • the "bait” construct contained an expression vector encoding a LexA-MyoD fusion protein.
  • pLexAMyoD57-166 To construct the "bait” plasmid, pLexAMyoD57-166, the MyoD insert from pEMSVMyoD ⁇ N ⁇ C (Weintraub et al. Genes Dev. 5: 1377-1386, 1991; which is inco ⁇ orated herein by reference in its entirety) was obtained by PCR.
  • the amplified fragment encoding amino acids 57-166 of MyoD was fused to the LexA DNA binding domain and used as a 'bait" by Ugation in-frame into the pBTM116 vector (Bartel et al., BioTechniques IA'. 920-924, 1993; which is inco ⁇ orated herein by reference in its entirety) to obtain a Polynucleotide sequence encoding a LexA-MyoD fusion wherein neither the amino nor the carboxyl terminal sequence of MyoD containing activation .domains were included.
  • Plasmid pBTMl 16 is a yeast 2 ⁇ M-bearing vector containing a LexA expression vector containing the ⁇ cerevisiae ADH promoter, the LexA DNA binding domain a polyUnker site and a terminator sequence containing a termination codon in all frames.
  • the S. cerevisiae Ade2 gene was isolated as a Bam HI fragment from pL909 plasmid (obtained from Ralph L. Keil, The Milton S.
  • Poly(A) + RNA was enriched by double passage through an oligo d(T) cellulose column.
  • First strand cDNA synthesis was achieved by reverse transcription of 100 ng of RNA at 42°C in 20 ⁇ l (microliters) of 500 pmol phosphorylated random hexamer and 50 U of Moloney murine leukemia vims reverse transcriptase (New England Biolabs) according to the manufacturer's instructions.
  • Second strand synthesis was carried out using E. coli DNA polymerase I, E. coli DNA Ugase and RNaseH.
  • the resulting cDNAs were blunt ended with T4 DNA polymerase and Pol I (Klenow). After enzyme inactivation and DNA precipitation, the cDNA was Ugated overnight with Not I adapters.
  • Complementary cDNAs m the range of 350 to 700 nucleotides were purified by agarose gel electrophoresis.
  • the cDNAs were amplified by PCR using primers corresponding to the Not I adapter.
  • the amplified cDNAs were digested with Not I, purified by agarose gel electrophoresis and Ugated into dephosphorylated, Not I-Unearized pVP16 (described by HoUenberg et al., ibid.).
  • the VP16 expression cassette in plasmid pVP16 contains, in order, a 1.5 kb S.
  • cerevisiae ADH1 promoter fragment and SP6 promoter, an initiator ATG, a nuclear localization signal, a VP16 activation domain, a Bam HI-Kpn I-Not-I polyUnker and termination codons in aU frames, and S. cerevisiae ADH1 termination sequences.
  • the two-hybrid screen was performed as described previously (Vojtek et al., Cell 74: 205-214, 1993 and HoUenberg et al., Mol. Cell. Biol. 15: 3813-3822, 1995; which are inco ⁇ orated by reference herein in their entirety) with some modifications.
  • the S. cerevisiae strain L40 containing muhimerized LexA binding sites cloned upstream of two reporter genes, the HIS3 gene and the ⁇ -galactosidase gene which were each integrated into the L40 genome was used as the host strain for the screen.
  • the host strain was transformed with the pLexAMyoD57-166 and subsequently transformed with the fusion Ubrary.
  • Transformants were selected in medium lacking histidine, and the histidine-positive ceUs were assayed for ⁇ -galactosidase activity.
  • the His ⁇ LacZ " * " clones were plated out in cloning density on synthetic plates lacking leucine and containing 0.01 g/1 of adenine. A single colony carrying only library constructs, indicated by its uniform red color, was selected from each clone for further analysis.
  • Selected yeast clones were mated with AMR70 strain carrying LexA fusions containing lamin, the bHLH of daughterless (Da) or Thingl (Thl) (HoUenberg et al., ibid.) to identify false positive clones and encoding Id or E protein fa ⁇ ly members, respectively.
  • the clones encoding members of the Id and E protein famiUes were further confirmed by sequence analysis. From approximately 8 x 10 ⁇ clones screened, 550 clones were selected that showed specific interaction with the MyoD bait.
  • I-mfb and I-mfc FuU length I-mf cDNA clones were obtained by screening an E10 and an El 6 mouse embryonic Ubrary (Novagen, Madison, WI) using an I-mf cDNA fragment isolated from the two-hybrid screen as probe. One clone was isolated from a screen of 1 x IO 6 plaques from the El 0 Ubrary. This clone was termed I-mfa. From 2 x 10* plaques screened from the El 6 Ubrary, two identical clones, designated I-mfb, and one I-mfc clone were isolated.
  • DNA sequencing and genomic mapping revealed that these three different transcripts are generated through differential poly-(A) adenylation and alternative splicing.
  • the genomic organization of I-mf was analyzed from two overlapping genomic clones ⁇ l and ⁇ 2 covering 31.9 kb genomic sequences. Mapping demonstrated that the I-mf gene contains five exons, and that I-mfa, I-mfb and I-mfc are alternative spUcing products of I- mf ( Figure).
  • the nucleotide sequences and deduced amino acid sequences of I-mfa, I-mfb and I-mfc are shown in SEQ ID NOS:l and 2, 3 and 4, and 5 and 6, respertively.
  • the deduced amino acid sequences suggested that the three I-mf proteins share a common amino terminal region, but each has a different carboxyl terminus.
  • the I-mfa and I-mfb carboxyl termini were approximately equal in size, and comprise one third of each polypeptide.
  • the third polypeptide, I-mfc had a shorter unique carboxyl terminus.
  • GenBank Database searches of the sequences of I-mf cONA and deduced I-mf amino acid sequences revealed no homology with any known cDNA or protein.
  • a search of the EST database identified a human fetal-lung cDNA that had high sequence similarity.
  • CCD disease cleidocranial dysplasia
  • a modified yeast one-hybrid system employing a ⁇ -galactosidase reporter was used to determine whether the fuU length I-mf and MyoD family members interact.
  • Constructs containing fuU length mouse MyoD, My ⁇ and Myogenin cDNAs were constructed by cloning PCR amplified protein coding regions of mouse MyoD, Myf5 and Myogenin cDNA, respectively, into yeast expression vector pEMBLye30/2 (Banroques et al., Cell 46: 837-844, 1986; which is inco ⁇ orated herein by reference in its entirety) to obtain plasmids pEMBLMyoD, pEMBLMyf.
  • Plasmid pEMBLMyoD containing full length MyoD with its own transactivation domain was co-transformed with constructs containing the LexA DNA binding domain fused to fuU length I-mfa, I-mfb or I-mfc (LexA- I-mfa, LexA-I-mfb, LexA-I-mfc, respectively) into yeast strain L40 by the standard lithium acetate method.
  • the deduced protein coding regions of I-mfa, I-mfb and I-mfc were amplified by polymerase chain reaction to prepare fragments suitable for subcloning. Briefly, the deduced protein coding region of I-mfa was ampUfied using the sense primer of SEQ ID NO: 7 (ACGAATTCCC AGG CCG ATG TCC CAG) and the antisense primer of SEQ ID NO: 8 (GTGCTCGAG CAC CCC ATG GCA TCA GGG).
  • the PCR product was _
  • the coding region of I-mfb was amplified and subcloned as described using the sense primer of SEQ ID NO: 7 and the antisense primer of SEQ ID NO: 9 (GTCCTCGAG GAC AAC TTA TTG GAG TTA) to generate pCS-I-mfb and pCSHA-I-mfb, respectively.
  • the coding region of I-mfc was ampUfied and subcloned as described using the sense primer of SEQ ID NO: 7 and the antisense primer of SEQ ID NO: 10 (TAGCTCGAG CTA ACT GGT TCT GTC CTA) to generate pCS-I-mfc and pCSHA-I-mfc, respectively.
  • Plasmid pCS2HA is a derivative of pCS2 that contains a 99 base pair insert encoding two haemaglutinin epitopes at the Cla I site of pCS2.
  • plasmids pCS-I-mfa and pCS-I-mfb were each digested with Eco RI and Hind HI to obtain the I-mf coding sequences. Each I-mf fragment was blunt ended. The I-mfa fragment was inserted into Bam Hl-linearized, blunt-ended pBTM116.
  • a plasmid containing the I-mfa in the correct orientation relative to the promoter was designated pLexA-I-mfa.
  • the I-mfb fragment was inserted into Eco Rl-Unearized, blunt- ended pBTMl 16.
  • a plasmid containing the I-mfb in the correct orientation relative to the promoter was designated pLexA-I-mfb.
  • plasmid pCS-I-mfc was digested with Ppu MI and Hind HI to obtain the I-mfc coding sequence. The fragment was blunt-ended and inserted into Bam HI, blunt-ended pBTMl 16.
  • a plasmid containing the I-mfc insert in the correct orientation relative to the promoter was designated pLexA-I-mfc.
  • ⁇ -galactosidase activities were determined quantitatively on exponential phase L40 yeast ceUs transformed with pEMBLMyoD, pEMBLMyf.
  • pLexA, pLexA-I-mfa, pLexA -I-mfb, pLexA-I-mfc or pLexA-DA encoding a fusion protein containing LexA and a bHLH domain of Daughierless, the Drosophila homologue of mammalian E proteins
  • pLexA-DA encoding a fusion protein containing LexA and a bHLH domain of Daughierless, the Drosophila homologue of mammalian E proteins
  • ceU peUets from 2 ml overnight cultures of each yeast transformant were suspended in 1 ml of Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KC1, 1 mM MgSO 4 , at pH 7.0) containing 3.5 ⁇ l of ⁇ -mercaptoethanol and the ODeoo was measured.
  • Cells were disrupted by adding 10 ⁇ l of chloroform and 10 ⁇ l of 0.1% sodium dodecyl sulfate foUowed by vigorous vortexing for 15 minutes.
  • AU of the LexA fusion proteins of appropriate size were expressed at similar levels when assayed by Western blot.
  • Table 1..L40 expressing LexA, LexA-I-mfa, LexA-I-mfb or LexA-I-mfc exhibited background levels of ⁇ -galactosidase activities ranging from 0.1 to 0.9 (arbitrary units). However, ⁇ -galactosidase activity was 5-fold higher than the LexA-I-mfa control in the same yeast strain bearing LexA-I-mfa and MyoD.
  • L40 expressing MyoD and LexA-I-mfb or LexA-I-mfc exhibited only background levels of ⁇ -galactosidase activity, indicating that MyoD interacted specificaUy with I-mfa in this assay, but not with I-mfb or I-mfc.
  • the transactivation potency of aU bHLH activators tested was also comparable to each other as assayed by the interaction with LexA-DA.
  • the MyoD sequence used in the original isolation of I-mf is conserved among myogenic factors suggesting that I-mf may interact "with other MyoD family members.
  • the interaction between I-mf and other MyoD fa ⁇ ly members was determined using the same one-hybrid system described above using pEMBLMyf. and pEMBLMyogenin. Using this system an interaction between I-mf and MyoD family members was detectable.
  • yeast co-expressing LexA-I-mfa and full length Myf5 or myogemn ⁇ -galactosidase activity was about 70- to 80-fold higher than in those expressing LexA-I-mfa alone.
  • a Ppu Ml/Hind III fragment of I-mfc cDNA encoding fuU length I-mfc was cloned in-frame into pMAL-c2 to generate pMBP-I-mfc.
  • An Eco O109I fragment of I-mfb cDNA, which contains amino acids 164 to 251 of I-mfb was cloned in-frame into pMAL-c2 to generate pMBP-I-mfb ⁇ N.
  • MBP-I-mf fusion proteins were purified using a bacterial expression system as described (Vojtek et al., ibid.). Briefly, bHLH proteins (Myf5, MyoD, Myogenin and E12) were synthesized in vitro using a TNT-coupled reticulocyte system (Pr omega Co ⁇ oration, Madison, WI) and labeled with 35s_ m ethionine, according to the instrurtions of the manufacturer.
  • an immunoprecipitation assay was performed on extracts from cells transiently co-transfected with pEMSVMyoD and an expression vector encoding haemaglutinin either (HA) epitope-tagged I-mfa (HA-I-mfa), HA-I-mfb or HA-I-mfc.
  • Expression vectors containing fuU length MyoD, Myf5 and Myogenin were constructed by subcloning the PCR ampUfied protein coding region of MyoD, Myf5 or Myogenin into the mammalian expression vector pEMSVscribe (Davis et al., Cell ii: 987- 1000, 1987; which is inco ⁇ orated herein by reference in its entirety) to create pEMSVMyoD, pEMSVMyf. or pEMSVMyogenin.
  • NIH3T3 ceUs were co- transfected with pEMSVMyoD and either pCSHA-I-mfa, pCSHA-I-mfb, or pCSHA-I-mfc.
  • Protein complexes were eluted with SDS protein sample buffer prior to analysis by Western blotting using peroxidase conjugated anti-HA (12CA5) antibody (Boehringer Mannheim, Indianapolis, IN) and the ECL detection system (Amersham Life Science, ArUngton Heights, IL).
  • Example 4 Interaction with Specific Domains of MyoD
  • LexA fusion constructs containing a series of MyoD deletions were made by cloning the PCR products of MyoD mutants from their corresponding pEMSV plasmids (Weintraub et al., Genes Dev. 5: 1377-1386, 1991; which is inco ⁇ orated herein by reference.
  • MyoD deletion mutant sequences in DM3-56 TM167, DM3-74/TM167, DM3-92/TM167, DM4- 101/TM167 and DM4-140/TM167 were amplified using oligonucleotide primers of SEQ ID NO: 11 (CTGAATTCC AGG AAC TGG GAT ATG) and of SEQ ID NO: 12 (GTTGGATCC TCA AAG CAC CTG ATA AAT CG), which hybridized to flanking sequence common to all the plasmids.
  • the PCR products were cloned in-frame into plasmid pBTM116.
  • the Pml I/Stu I, Pml I/Sty I, Pml I/Nar I or Pml I Mlu I fragments were cloned in-frame into pBTM116 to generate LexA-MyoD:dDM53/127, dDMD53/146, dDM53/173 and dDM53/199 respertively.
  • Table 2 shows the LexA-MyoD mutant fusion proteins and the amino acids of MyoD that are deleted.
  • VP16 fusion proteins containing the full length I-mfa in the L40 yeast strain ⁇ - Galactosidase levels were determined to measure the interaction between I-mfa and the MyoD mutants.
  • Helbc 2 of MyoD was dispensable for this interaction; however it was required for dimerization with bHLH proteins.
  • the bHLH domain of MyoD was sufficient for dimerization with bHLH proteins; however, it was not sufficient for interaction with I-mfa.
  • a series of LexA fusion constructs were prepared containing deletion mutants of I-mfa.
  • the deletion mutant fusions were constructed by cloning the corresponding PCR products of I-mfa in-frame into pBTMll ⁇ .
  • Oligonucleotide primers of SEQ ID NO: 13 ACGGAT CCC AGG CCG ATG TCC CAG
  • SEQ ID NO: 14 CTCTCGAGTCA GCA GCA GCA GAG GCA GGA G
  • the resulting fragment was subcloned in-frame into pBTM116 such that the resulting vertor encodes a LexA-I-mf fusion or in-frame into pCS2HA such that the resulting vector encodes an HA-I-mf.
  • OUgonucleotide primers of SEQ ID NO: 15 ACGGAT CCC CAG ACC ATG TCC CTC CTC
  • SEQ ID NO: 16 GTGCTCGAG CAC CCC ATG GCA TCA GGA
  • Oligonucleotide primers of SEQ ID NO: 17 (CAGAATTCA CAG CCT CAA GGG AAC CCC) and of SEQ ID NO: 16 were used to amplify the region encoding I-mfa amino acids 87-246, and the PCR product was subcloned into pBTMl l ⁇ and pCS2HA as described above.
  • Oligonucleotide primers of SEQ ED NO: 18 (GTGAATTCA GAT TGC TGC GTC CAC TGC) and of SEQ ID NO: 16 were used to amplify the region encoding I-mfa amino acids 163-246, and the PCR product was subcloned into pBTMl l ⁇ and pCS2HA as described above.
  • OUgonucleotide primers of SEQ ID NO: 19 (GTGAATTCC TGC TGT GGG TCC GGC GAG) and of SEQ ID NO: 16 were used to amplify the region encoding I-mfa amino acids 203-246, and the PCR product was subcloned into pBTMl l ⁇ and pCS2HA as described above.
  • OUgonucleotide primers of SEQ ID NO: 17 and of SEQ ID NO: 14 were used to amplify the region encoding I-mfa amino acids 87-202, and the PCR product was subcloned into pBTMl l ⁇ and pCS2HA as described above.
  • Oligonucleotide primers of SEQ ID NO: 18 and of SEQ ID NO: 14 were used to amplify the region encoding I-mfa amino acids 163-202, and the PCR product was subcloned into pBTMll ⁇ and pCS2HA as described above.
  • the LexA-I-mfa fusion mutants were co-transformed with pEMBLMyf5 into the yeast strain L40.
  • the interaction between each fusion mutant and Myf5 was indicated by ⁇ -Galactosidase activity which was assayed as described herein.
  • the region of I-mfa common to all three I-mf proteins (amino acids 1-162 of SEQ ID NOS:2, 4, and 6) was not required for interaction with Myf5 as suggested by the high level of ⁇ -Galactosidase expressed in ceUs co-expressing Myf5 and LexA-I-mfa(163-246).
  • the carboxyl-terminal half of the domain unique to I-mfa was also dispensable for this interaction, as deduced from the level of ⁇ -Galartosidase in cells co-expressing My£5 and LexA-I-mfa( 1-202).
  • the smallest interactional domain that retained activity among those deletion mutants tested was located in a 40 amino acid segment (from amino acids 163-202 of SEQ ID NO: 2) encompassing the amino-terminal half of the unique domain of I-mfa.
  • the LexA-I-mfa fusion mutants were also co-expressed with VP16-I-mfa, VP16-I- mfb, or VP16-I-mfc.
  • amino acids 87-163 of SEQ ID NO: 2 from the common region of I-mfa was able to enhance the self-interaction as determined by comparing the results of cells co-expressing VP16-I-mfa and LexA-I- mfa(87-246) or LexA-I-mfa(163-246) and of cells co-expressing VP16-I-mfa and LexA-I- mfa(87-202) or LexA-I-mfa(163-202) (Table 3).
  • LexA-I-mfb ⁇ 0.1 0.2 ⁇ 0.1 ⁇ 0.1 ⁇ 0.1
  • LexA-I-mfc ⁇ 0.1 ⁇ 0.1 ⁇ 0.1 ⁇ 0.1 ⁇ 0.1 ⁇ 0.1 ⁇ 0.1
  • Plasmid p4R-TK-CAT is a modification of the plasmid TK-CAT in which four oligomerized MyoD binding sites are inserted upstream of TK.
  • CAT artivity levels were determined for the transfected cells as described above (Gorman et al., Mol. Cell. Biol. 2: 1044-1051, 1982; which is inco ⁇ orated herein by reference in its entirety).
  • the CAT activities were normalized to the value from Myf5 or E12 co-transfected with pCS2HA, which were set to 100 (Table 4).
  • the minimal interactional domain of I-mfa (amino acids 163-202 of SEQ ID NO: 2) repressed 55% of the My£5 transactivation activity.
  • the larger polypeptide (amino acids 87-202 of SEQ ID NO: 2) demonstrated 93% inhibition.
  • Western blot analysis demonstrated that the minimal interactional domain of I-mfa (amino acids 163-202 of SEQ ID NO: 2) was less stable than the larger polypeptide (amino acids 87-202 of SEQ ID NO: 2).
  • I-mfa associates more weakly with MyoD than with other myogenic regulatory factors in mouse embryos. I-mfa also had sUghtly weaker effects on MyoD than on the other myogenic factors tested in both transactivation repression and cytoplasmic retention assays.
  • Example 5 I-mfa Function in Myogenesis To investigate the possible function of I-mf in embryonic myogenesis, the expression pattern of I-mfa during mouse embryogenesis was determined by in situ hybridization as generaUy described by Lee et al. (in commonly assigned, co-pending international patent appUcation WO 95/30693; and Science 268: 835-844, 1995; which are inco ⁇ orated herein by reference in their entirety) and Hurlin et al. (EMBO J. 14: 5646- 5659, 1995; which is inco ⁇ orated herein by reference in its entirety). Briefly, mouse embryos at El 1.5 from inbred strain BL.6 were coUected for RNA in situ hybridization. The embryos were staged such that the day of the vaginal plug was considered as embryonic day 0.5 (E0.5). The embryos were fixed, paraffin embedded and sectioned before hybridization.
  • I-mfa transcripts were highly expressed in the sclerotome. However, the expression of MyoD was concentrated in the myotome. No specific hybridization was found when the corresponding I-mfa sense riboprobe was used in control in situ hybridization.
  • the transverse sections of caudal somites of the same stage embryos showed high level of I-mfa expression in the sclerotome, the ventral subdomain of the somites, while Myf5 was detected exclusively in the myotome part of dermomyotome, the dorsal subdomain of somites.
  • a simUar expression pattern of I-mfa was also observed in newly formed somites ofE8.5 mouse embryos.
  • I-mfa In addition to its expression in somites, I-mfa was also shown to be expressed in the notochord and at lower levels in the neural tube as weU as in Umb buds, heart, branchial arches, head mesenchyme and various other parts of the mouse embryo. Moreover, analysis of RNA from various mouse tissues revealed that I-mfa was most highly expressed in skeletal tissue, but was expressed at lower levels in almost every other tissue analyzed. Early in development I-mfa was highly expressed in the sclerotomal domain of somites. In contrast, myogenic factors were detected in the dermomyotomal domain of somites.
  • Example 6 I-mfa Inhibits the MyoD Family-Dependent Transcriptional Activation
  • the ability of MyoD family members to transactivate an E box-driven reporter construct was assayed in the presence or absence of I-mf to determine if I-mf influences the artivity of myogenic factors. Transactivation was assessed by using the E.box-driven reporter construct, p4R- TK-CAT (Weintraub et al., Genes Dev.
  • Expression vectors containing full length My£5 were constructed by subcloning the PCR amplified protein coding region of Myf5 into the mammalian expression vector pCS2NLS (Rupp et al., ibid., 1994) to create pCSNLSMyf. .
  • NIH3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum on 10 cm dishes.
  • DMEM Dulbecco's modified Eagle's medium
  • the ceUs were transfected using the calcium phosphate precipitation method (Davis et al., CeU 60: 733-746, 1990; which is inco ⁇ orated herein by reference) with 5 ⁇ g of the p4R-TK-CAT reporter construct and each possible combination of 5 ⁇ g of pEMSVMyf. or pEMSVMyogenin and 5 ⁇ g of either pCSHA-I-mfa, pCSHA-I-mfb, or pCSHA-I-mfc.
  • every combination of 3 ⁇ g of each I-mf vertor and 3 ⁇ g of each myogenic expression plasmid were transferted into cells on 6 cm plates using the calcium phosphate precipitation method. The following day, the cells were rinsed with PBS and incubated for 10 hours in DMEM plus 10% calf serum. After the incubation, the cells were switched into differentiation medium, DMEM plus 2% horse serum, for an additional 40 hours. Cells were harvested and CAT assays were performed as described above or fixed for indirect immunofluorescence staining.
  • Indirect immunofluorescence staining was carried out on transfected ceUs that were fixed in PBS with 4% paraformaldehyde for three minutes and permeabilized in PBS with 0.25% Triton X-100 for three minutes.
  • the fixed ceUs were stained with anti-Myf5 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-myosin heavy chain monoclonal antibody MF20 (Boehringer Mannheim, IndianapoUs, IN) or anti-HA monoclonal antibody, 12CA5 (Boehringer Mannheim, IndianapoUs, IN).
  • I-mfa reduced the transactivation activity of all three myogenic factors tested to 10 to 23% of the control level (Table 5).
  • I-mfb and I-mfc showed no significant effect on the myogenic fartor-dependent reporter activation.
  • I-mfa did not significantly inhibit the transactivation artivity of El 2, when pCS- I-mfa and an analogous E12 construct were co-transfected with the p4R-TK-CAT reporter construct (Table 5).
  • the relative value of 100% in MyoD, My£5, Myogenin and E12 plus control vector transferted ceUs represented absolute CAT values of 55,400, 53,070, 17,430 and 53,570, respectively. Comparable expression levels and appropriate size I-mfa, I-mfb and I-mfc were confirmed by Western blot analysis.
  • I-mfa activity of repressing myogenic regulatory factor activity occurs in a dose-dependent manner.
  • MyoD and Myf5 were each transiently co-expressed with either I-mfa, I-mfb or I- mfc in mouse C3H10T1/2 cells (C3H/10T1/2; Accession No. CCL 226, American Type Culture Collection, 12301 Parklawn Drive, RockviUe, MD 20852) containing p4R-TK- CAT.
  • the CAT activity from the co-transfertions were normalized to the value from each co-transfection with empty vertor to allow direct comparison of I-mf artivity associated with each myogenic fartor.
  • I-mfc 100 106 111 100 101 MyoD, MyfS and Myogenin were each transiently co-expressed with either I-mfa, I- mfb or I-mfc in NIH3T3 cells containing p4R-TK-CAT.
  • the CAT activity was normalized as described previously.
  • the 100% value in MyoD, MyfS and Myogenin plus control vertor transfected NIH3T3 ceUs were 37,168, 79,532 and 32,968, respertively.
  • I-mfa Inhibits the Mvf5-Mediated Muscle Differentiation
  • I-mfa and Myf5 were co-expressed transiently in NIH3T3 fibroblasts as described above, and muscle differentiation was assayed by the appearance of the specific muscle marker myosin heavy chain (MyHC), as weU as by cellular mo ⁇ hology.
  • MyHC myosin heavy chain
  • the cells were co-transfected with pEMSVMy£5 and either the control vertor pCS2, pCS-I-mfa, pCS-I-mfb or pCS-I- mfc.
  • cells Twenty-four hours after transfection, cells were ⁇ f grown in differentiation medium for an additional 40 hours before staining. After incubation in the differentiation medium, the cells were fixed in PBS with 4% of paraformaldehyde for 3 minutes and permeabUized in PBS with 0.25% Triton X-100 for 3 minutes.
  • the fixed cells were double stained with a Myf5 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) and the anti-myosin heavy chain monoclonal antibody, MF20 followed by incubation with fluorescein (DTAF)- conjugated donkey anti-rabbit Ig and Rhodamine (TRITC)-conjugated goat anti-mouse Ig (Jackson ImmunoResearch Laboratories, West Grove, PA). The images of the labeled cells were superimposed.
  • DTAF fluorescein
  • TRITC Rhodamine
  • Example 8 I-mfa Retains MyoD Family Members in the Cytoplasm by Masking their Nuclear Localization Signals
  • NIH3T3 ceUs were co-transfected with ⁇ EMSVMyf5 and either pCSHA-I-mfa, pCSHA-I- mfb or pCSHA-I-mfc.
  • Indirect immunofluorescence staining was performed as described above using an anti-MyfS polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA) and the anti-HA monoclonal 12CA5 (Boehringer Mannheim, IndianapoUs, IN) antibodies.
  • I-mfa protein was distributed primarily throughout the cytoplasm, although weak staining was detectable in the nucleus. This subceUular localization did not change in the presence of MyfS. In the absence or in the presence of I- mfb or I-mfc, Myf5 was localized exclusively in the nucleus. In contrast, when co- expressed with I-mfa, MyfS protein co-localized with I-mfa in the cytoplasm.
  • Co-localization assays were also carried out on cells co-expressing I-mfa and MyoD, Myogenin or Myc-epitope-tagged E47.
  • NIH3T3 ceUs were transiently transfected with pCSHA-I-mfa and either pEMSVMyoD, pEMSVMyogenin or pEMSVE47.
  • the corresponding cells were immunostained with polyclonal MyoD antiserum, Myogenin monoclonal antibody, F5D (obtained from Dr. Wood Wright, University of Texas Southwestern Medical Center, Dallas, TX; Cusella-DeAngelis et al., Cell Biol.
  • MyoD has two nuclear localization signals (NLS), one in the basic region and one in the helbc 1 domain (Vandromme et al., Proc. Natl. Acad. Sci. USA 92: 4646-4650, 1995).
  • NLS nuclear localization signals
  • the location of these NLS overlaps with the I-mf interaction region, raising the possibility that I-mfa may simply mask the NLS of the MyoD family members, resulting in their cytoplasmic retention. If this masking hypothesis were correct, adding a presumably unmasked NLS outside of the C/H rich and bHLH regions of myogenic regulatory factors should restore their nuclear localization.
  • an expression plasmid was constructed containing one SV40 T antigen NLS added to the amino terminus of MyfS, designated pCS2NLSMy£5. This construct was co-transfected with pCSHA-I-mfa and visualized by indirect immunostaining as described previously. When co-expressed with I- mfa in NIH3T3 ceUs, NLSMyf5 localized exclusively in the nucleus.
  • I-mfa The in vitro binding of I-mfa to NLSMyogenic factors was assessed. Briefly, plasmids pEMSVMyfS, pEMSVMyogenin, pEMSVMyoD, pCS2NLSMyf5 and pCS2NLSMyogenin were in vitro translated as described previously. The labeled proteins were divided and one portion was subjected to co-precipitation with amylose resin following incubation with the purified Maltose Binding Protein-I-mf fusion, MBP-I-mfa ⁇ N, which contains amino acids 163 to 246 of I-mfa as previously described. SDS- polyacrylamide gel electrophoresis on the in vitro and co-precipitated samples demonstrated that I-mfa associates as a binding partner equally weU with NLSMyf5 and My ⁇ .
  • NIH3T3 ceUs transiently transfected with MyfS and I-mf constructs were examined.
  • NIH3T3 were transiently transfected with pCS2NLSMyf5 and either pCSHA-I-mfa or pCSHA-I-mfc as described previously. Twenty-four hours post-transfertion, cells were grown in differentiation medium for an additional 40 hours before they were stained with antibodies.
  • Double immunostaining was performed with MyfS antiserum (Santa Cruz, Santa Cruz, CA) and the anti-MyHC monoclonal antibody, MF20 (Boehringer Mannheim), foUowed by staining with Fluorescein conjugated anti-rabbit Ig antibody and Rhodamine conjugated anti-mouse Ig antibody, respertively. The staining images were superimposed. Expression of NLSMyf5 alone induced MyHC expression in transferted NIH3T3 ceUs to a simUar extent as wild type Myf5.
  • the DNA binding activity of E47 was not affected by I-mfa ⁇ N even when present in a 192 fold molar excess, indicating that I-mfa interferes specifically with DNA binding of myogenic factors.
  • Knock-out mice in which parts of the murine I-mf coding sequence was replaced with the neomycin resistance gene (neo) were generated to assess the consequences of eliminating the murine I-mf protein during mouse development.
  • Genomic I-mf sequences used for these knock-out mice were obtained from the 129/Sv mice so the homologous recombination could take place in a congenic background in 129/Sv mouse embryonic stem ceUs.
  • I-mf genomic clones were isolated from a genomic Ubrary prepared from 129/Sv mice (Zhuang et al., Cell 79: 875-884, 1994; which is inco ⁇ orated herein by reference in its entirety) using a random-primed I-mfa cDNA probe.
  • Plasmid pPNT (Tybulewicz et al., Cell 65: 1153-1163, 1991; which is inco ⁇ orated herein by reference in its entirety) provided the vertor backbone for the targeting construct.
  • Plasmid pPNT contains the Herpes simplex virus thymidine kinase gene (hsv-tic, a negative selection marker) under the control of the PGK promoter neomycin resistance gene (neo; a positive selection marker) under the control of the PGK promoter such that the PGK-neo expression cassette is in the opposite orientation relative to the PGK-TK expression cassette.
  • hsv-tic Herpes simplex virus thymidine kinase gene
  • neomycin resistance gene neo; a positive selection marker
  • pPNT- ⁇ I- mfa a 2.7 kb Pst I-Kpn I 5' I-mf genomic fragment containing 5' intron sequences and a portion of the coding region of exon IV was cloned between the PGK-TK and the PGK- Neo cassettes and a 3.2 kb Mlu I-Kpn I 3' I-mf genomic fragment containing a portion of the coding region of exon V and 3' non-coding sequences was inserted downstream of the PGK-neo cassette.
  • the resulting plasmid, pPNT- ⁇ I-mfa contained a replacement of a 5 kb region of the I-mf gene with the PGK-neo expression cassette.
  • the targeting construct was linearized at a unique restriction site in the vector backbone and was transfected by electroporation into mouse embryonic stem (ES) cells.
  • ES mouse embryonic stem
  • a 129/Sv derived ES ceU Une, AK-7 described by Zhuang et al. (ibid.) was used for electroporation.
  • ES cells were routinely cultured on mitomycin C-treated (Sigma) SNL 76/7 cells (feeder cells) as described by McMahon and Bradley (Cell ⁇ 2: 1073-1085, 1990; which is inco ⁇ orated herein by reference in its entirety) in culture medium containing high glucose DMEM supplemented with 15% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT) and 0.1 ⁇ M (micromolar) ⁇ -mercaptoethanol.
  • 25 ⁇ g (micrograms) of the targeting construct was linearized by digestion with Not I, phenol-chloroform extracted, and ethanol precipitated.
  • the linearized vertor was then elertroporated into 1-2 x IO 7 AK-7 (ES)ceUs.
  • the electroporated ceUs were seeded onto five 10-cm plates. After 24 hours, G418 was added to each of the plates to a final concentration of 200 ⁇ g/ml (micrograms per mill Uiter). After three days gancyclovir was added to four of the plates to a final concentration of 2 ⁇ g/ml (micrograms per milUliter). This selertion enriches the population of ES cells that have undergone homologous recombination by eliminating ES ceU clones that have randomly integrated the targeting construct.
  • primers NK2 and AC48 CATCTAGGAATGAAGTAAGCAGGA (SEQ ID NO:20) and GCAAGTTGGTGATGCTGTCTGTCTGTGGATGCG (SEQ ID NO:21), respertively) were used in the PCR reaction.
  • primers NK3rev and YZ29 AATCAATGTGCCTCCTAGATCTCTAGCC (SEQ D NO:22) and TCGCAGCGCATCGCCTTCCTA (SEQ ED NO:23), respertively) were used in the PCR reaction.
  • each positive clone was thawed and passaged once on feeder ceUs.
  • the transferted ceUs were trypsinized into single cells, and blastocysts obtainedlrom C57BL 6J mice were injected with approximately 15 cells.
  • the injected blastocysts were then implanted into pseudopregnant mice (C57BL 6J x CBA).
  • Five male and 6 female chimeras arose from the injected blastocysts.
  • One male chimera and one female chimera gave germ-line transmission at a high rate as determined by the frequency of agouti coat color transmission to their offspring (Fl) in a cross with C57BL 6J female mice.
  • genomic DNA prepared from taU biopsies was digested with Hind DI and Xba I and probed with probe specific to exon DI of I-mf. This probe detects a 17 kb fragment from the wild-type allele and a 7 kb fragment from the mutant allele. Therefore, a Southern analysis would show a single 13 kb band for a wild-type mouse, 13 kb and 7 kb fragments for a heterozygous mouse, and a single 7 kb band for a homozygous mutant mouse.
  • heterozygous (+/-) mice were mated with sibling heterozygous mice to give rise to the homozygous (-/-) mutant mice.
  • Heterologous (+/-) mice were kept on a hybrid C57BL 6J x 129 background, were back-crossed once to give inbred 129 mice or were back-crossed multiple times into a C57BIJ6J background. The majority of homozygous mutant mice in the C57BI76J background die during embryogenesis.
  • ORGANISM Mus musculus
  • CAGAATCCCA ACCCAGATGT GAGAAGGTGG GACGCTCAGA GGGGCCACCT CAGCCACCGA 898
  • Leu Thr Leu Cys Asn lie Leu Leu Asp Cys Ala Thr Cys Gly Ser Cys 180 185 190
  • ORGANISM Mus musculus
  • ATC CAC AGA CAG ACA GCA TCA CCA ACT TGC TGC TTA AGA AAT GCA CAA 580 He His Arg Gin Thr Ala Ser Pro Thr Cys Cys Leu Arg Asn Ala Gin 170 175 180
  • ORGANISM Mus musculus

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Abstract

On décrit un nouveau gène, inhibiteur de la famille MyoD (I-mf), lequel code des nouvelles protéines, I-mfa, I-mfb et I-mfc, impliquées dans la régulation de la myogenèse, lors du développement des vertébrés. I-mf présente un fort taux d'expression dans le sclérotome de vertébrés en cours de développement et est censée jouer un rôle important dans la modélisation du somite et la détermination du sort de la cellule sclérotomale. Un domaine unique d'interaction C-terminal de la protéine I-mf induit des interactions physiques entre I-mfa et des membres de la famille MyoD d'activateurs de transcriptions et agit pour inhiber la transactivation de gènes spécifiques des muscles, provoquée par des membres de la famille MyoD, effectuant ainsi une répression de la myogenèse. Une autre caractéristique de l'activité de I-mf consiste en ce que I-mf s'associe à des protéines membres de la famille MyoD et retient celles-ci dans le cytoplasme en masquant les signaux de localisation nucléaire de ces protéines membres. Sur la base de ces gènes et protéines I-mf ici décrits, on a mis au point plusieurs procédés et compositions de criblage, d'isolement et de caractérisation de facteurs endogènes et exogènes, de médicaments et d'agents thérapeutiques utiles pour évaluer et/ou réguler le développement normal et anormal de la myogenèse, et la différenciation cellulaire.
PCT/US1997/014780 1996-08-27 1997-08-21 Nouvelles sequences polynucleotidiques codant des proteines impliquees dans la myogenese WO1998008860A1 (fr)

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US20040158289A1 (en) * 2002-11-30 2004-08-12 Girouard Steven D. Method and apparatus for cell and electrical therapy of living tissue
US7840263B2 (en) * 2004-02-27 2010-11-23 Cardiac Pacemakers, Inc. Method and apparatus for device controlled gene expression
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